Depletion of luminal iron alters the gut microbiota and prevents Crohn's disease-like ileitis

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DOI: 10.1136/gut.2010.216929 · Source: PubMed
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Abstract
Iron replacement therapy is a common treatment in patients with anaemia and Crohn's disease, but oral iron supplements are less tolerated. The pathogenesis of Crohn's disease is attributed to intestinal bacteria and environmental factors that trigger disease in a genetically predisposed host. The aim of this study was to characterise the interrelationship between luminal iron sulfate, systemic iron, the gut microbiota and the development of chronic ileitis in a murine model of Crohn's disease. Wild type (WT) and heterozygous TNF(ΔARE/WT) mice were fed with an iron sulfate containing or iron sulfate free diet in combination with intraperitoneal control injections or iron injections for 11 weeks. TNF(ΔARE/WT) mice develop severe inflammation of the distal ileum but remained completely healthy when transferred to an iron sulfate free diet, even if iron was systemically repleted. Absence of luminal iron sulfate reduced cellular markers of endoplasmic reticulum (ER) stress responses and pro-apoptotic mechanisms in the ileal epithelium. Phenotype or reactivity of major effector intraepithelial CD8αβ(+) T cells were not altered in the absence of luminal iron. Interestingly, ER stress mechanisms sensitised the small intestinal epithelial cell (IEC) line Mode-K to cytotoxic function of effector T cells from TNF(ARE/WT) mice. Pyrosequencing of 16S rRNA tags of the caecal microbiota revealed that depletion of luminal iron sulfate induced significant compositional alterations, while total microbial diversity (Shannon's diversity index) and number of total operational taxonomic units were not affected. This study showed that an iron sulfate free diet in combination with systemic iron repletion prevents the development of chronic ileitis in a murine model of Crohn's disease. Luminal iron may directly affect IEC function or generate a pathological milieu in the intestine that triggers epithelial cell stress-associated apoptosis through changes in microbial homeostasis. These results suggest that oral replacement therapy with iron sulfate may trigger inflammatory processes associated with progression of Crohn's disease-like ileitis.
doi: 10.1136/gut.2010.216929
published online November 12, 2010Gut
Tanja Werner, Stefan J Wagner, Inés Martínez, et al.
ileitis
microbiota and prevents Crohn's disease-like
Depletion of luminal iron alters the gut
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Depletion of luminal iron alters the gut microbiota and
prevents Crohn’s disease-like ileitis
Tanja Werner,
1
Stefan J Wagner,
1
Ine
´s Martı
´
nez,
2
Jens Walter,
2
Jung-Su Chang,
1
Thomas Clavel,
1
Sigrid Kisling,
1
Klaus Schuemann,
3
Dirk Haller
1
ABSTRACT
Background Iron replacement therapy is a common
treatment in patients with anaemia and Crohn’s disease,
but oral iron supplements are less tolerated. The
pathogenesis of Crohn’s disease is attributed to intestinal
bacteria and environmental factors that trigger disease in
a genetically predisposed host. The aim of this study was
to characterise the interrelationship between luminal iron
sulfate, systemic iron, the gut microbiota and the
development of chronic ileitis in a murine model of
Crohn’s disease.
Methods Wild type (WT) and heterozygous TNF
D
ARE/WT
mice were fed with an iron sulfate containing or iron
sulfate free diet in combination with intraperitoneal
control injections or iron injections for 11 weeks.
Results TNF
D
ARE/WT
mice develop severe inflammation
of the distal ileum but remained completely healthy when
transferred to an iron sulfate free diet, even if iron was
systemically repleted. Absence of luminal iron sulfate
reduced cellular markers of endoplasmic reticulum (ER)
stress responses and pro-apoptotic mechanisms in the
ileal epithelium. Phenotype or reactivity of major effector
intraepithelial CD8a
b
+
T cells were not altered in the
absence of luminal iron. Interestingly, ER stress
mechanisms sensitised the small intestinal epithelial cell
(IEC) line Mode-K to cytotoxic function of effector T cells
from TNF
ΔARE/WT
mice. Pyrosequencing of 16S rRNA
tags of the caecal microbiota revealed that depletion of
luminal iron sulfate induced significant compositional
alterations, while total microbial diversity (Shannon’s
diversity index) and number of total operational
taxonomic units were not affected.
Conclusion This study showed that an iron sulfate free
diet in combination with systemic iron repletion prevents
the development of chronic ileitis in a murine model of
Crohn’s disease. Luminal iron may directly affect IEC
function or generate a pathological milieu in the intestine
that triggers epithelial cell stress-associated apoptosis
through changes in microbial homeostasis. These results
suggest that oral replacement therapy with iron sulfate
may trigger inflammatory processes associated with
progression of Crohn’s disease-like ileitis.
INTRODUCTION
Present aetiological theories of inammatory bowel
diseases (IBDs) imply a role of environmental
factors like smoking, luminal enteric bacteria and
trace elements like iron for the pathogenesis in
a genetically susceptible host but mechanistic
data are lacking.
1
One third of patients with
IBD develop iron deciency anaemia due to an
inappropriate intake or loss of iron affecting the
quality of life. Oral supplementations are common
alternatives for the treatment of iron deciency
and contain iron in the form of Fe(II) salts, such
as Fe(II) sulfate, Fe(III) polymaltose complexes
or haem iron polypeptides. However, oral iron
replacement therapy is poorly tolerated and may
even contribute to the inammatory processes and
tissue pathology in patients with IBD.
23
The intestinal epithelium represents a highly
selective barrier between the gut lumen and
underlying cells of the immune system, and intes-
tinal epithelial cells (IECs) must adapt to constant
changes in their environment by processing both
bacterial and host-derived immune signals.
4
An
emerging paradigm suggests that stress responses in
the endoplasmic reticulum (ER) contribute to the
development of chronic intestinal inammation
through mechanisms that promote the loss of
<Additional material, figures
and tables are published online
only. To view these files please
visit the journal online (http://
gut.bmj.com).
1
Chair for Biofunctionality, ZIEL e
Research Center for Nutrition
and Food Science, CDD eCenter
for Diet and Disease, Technische
Universita
¨tMu
¨nchen,
Freising-Weihenstephan,
Germany
2
Department of Food Science
and Technology, University of
Nebraska, 333 Food Industry
Complex, Lincoln, Nebraska,
USA
3
Molecular Nutrition Unit, ZIEL e
Research Center for Nutrition
and Food Science, Technische
Universita
¨tMu
¨nchen,
Freising-Weihenstephan,
Germany
Correspondence to
Professor Dirk Haller,
Biofunctionality, Technische
Universita
¨tMu
¨nchen,
Gregor-Mendel-Str. 2, 85350
Freising-Weihenstephan,
Germany; haller@wzw.tum.de
Accepted 22 September 2010
Significance of this study
What is already known about this subject?
<In animal models, a high-iron diet enhances
intestinal inflammation, and iron induces ROS
production and therefore DNA damage.
<In human trials, it is suggested that oral iron
enhances the inflammation index. Patients with
IBD often need iron supplementation.
<Endoplasmic reticulum (ER) stress is a possible
pathological factor in IBD.
What are the new findings?
<Oral iron deprivation prevents chronic ileitis in
TNF
D
ARE/WT
mice and systemic iron repletion
maintained the protective effect of luminal iron
deprivation.
<Iron triggers ER stress in intestinal epithelial
cells and ER stress sensitises the epithelium
towards cytotoxic T cell induced apoptosis.
<The comprehensive analysis of the caecal
microbiota by pyrosequencing of 16S rRNA
tags in WT and TNF
ΔARE/WT
mice revealed that
the depletion of luminal iron sulfate and not
inflammation per se induced significant compo-
sitional alterations of the caecal microbiota.
How might it impact on clinical practice in the
foreseeable future?
<These data imply that systemic iron repletion
should be preferred for the treatment of anaemic
patients with Crohn’s disease as orally admin-
istered iron sulfate led to intestinal inflammation
in susceptible TNF
D
ARE/WT
mice.
Werner T, Wagner SJ, Martı
´
nez I, et al.Gut (2010). doi:10.1136/gut.2010.216929 1 of 9
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tissue homeostasis including goblet
5
and Paneth cell dysfunc-
tions.
6
We showed that immune-mediated control mechanisms
modulate ER stress-associated activating transcription factor
(ATF) 6 signalling at the IEC level. The lack of immune
homeostasis triggered ER stress-dependent mechanisms for the
development of chronic intestinal inammation in animal
models and human IBD.
7
Consistent with these ndings, Brandl
et al identied an N-ethyl-N-nitrosourea (ENU)-induced muta-
tion that disrupts ATF6-driven ER stress responses leading to
increased susceptibility for dextran sodium sulfate (DSS)-
induced colitis in mice.
8
Most importantly, gene variants of the
transcription factor X-box protein-1 (XBP-1) were linked to an
increased risk for IBD.
6
Heterozygous TNF
D
ARE/WT
mice show impaired regulation of
tumour necrosis factor (TNF) synthesis and develop a severe
CD8
+
T cell-dependent ileitis closely resembling the immune-
and tissue-related phenotype of human Crohns disease with
ileal involvement.
9
Functional proteome analysis of primary ileal
IECs from wild-type (WT) and TNF
ΔARE/WT
mice fed an iron
sulfate containing diet or iron sulfate free diet revealed signi-
cant differences in the regulation of proteins participating in
energy metabolism and stress responses when comparing
inamed with non-inamed conditions.
10
In this study, we used
heterozygous TNF
ΔARE/WT
mice to investigate the mechanistic
role of both luminal iron sulfate and systemic iron in the
development of chronic ileitis in a model of Crohns disease
focussing on the effect of iron status on ER stress regulation and
changes in gut microbial ecology.
MATERIAL AND METHODS
Animals
Conventionally raised WT and heterozygous TNF
D
ARE/WT
mice
on a C57BL/6 background received an iron sulfate containing
(Altromin C1000, 180 mg Fe/kg as iron sulfate) or iron sulfate
free diet (Altromin C1038, <10 mg Fe/kg; N¼10/group) for
11 weeks after weaning period (age of 7 weeks). Mice were
housed in groups according to gender and treatment. At the age
of 18 weeks, mice were killed by cervical dislocation.
An initial dose nding study was performed to evaluate the
concentrations for systemic iron replacement with Fe(NO
3
)
3
complexed with nitrilotriacetic acid (NTA) in a 1:2 ratio [FeNTA
(1:2)]. The iron sulfate diet contained 180 mg/kg (180 mg/g)
iron sulfate and, based on an average daily intake of 720 mgof
iron sulfate, the mice were administered an average of 28 mg iron
sulfate/g mouse per day. Thebioavailability (BV) for iron sulfate is
estimated to be about 10% (2.8 mg Fe/g mouse), resulting in an
equivalent of 50 nmol [FeNTA(1:2)]/g mouse/day for the
systemic iron injection. By applying one injection per week, 10%
bioavailability corresponds to 360 mmol (350 mmol+10 mmol as
safety value) iron per week. We tested four concentrations of
FeNTA for the systemic iron repletion including 360 mmol (10%
BV), 180 mmol (5% BV), 90 mmol (2.5% BV) and 45 mmol (1.25%
BV). Due to the toxic effects of 360 mmol (10% BV) and 180 mmol
(5% BV) FeNTA concentrations, the mice received weekly intra-
peritoneal injections of 90 mmol FeNTA per g body weight to
restore the hepatic non-haem iron concentrations. Control mice
on the iron sulfate containing and iron sulfate free diet received
NaCl control (Ctrl) injections (0.9% NaCl per g body weight).
For the analysis of tissue pathology, tissue sections of the
distal ileum were xed in 10% neutral buffered formalin.
Histopathology was measured by blindly assessing the degree of
lamina propria mononuclear cell inltration, crypt hyperplasia,
goblet cell depletion and architectural distortion as previously
described.
11
Isolation and phenotyping of intraepithelial lymphocytes
See supplemental material
Isolation of primary mouse IECs
Primary IECs from ileal epithelium of WT as well TNF
ΔARE/WT
mice were puried as previously described.
12
Western blot analysis
Puried primary IECs (pooled for each group; equal amount of
protein used from each single animal) or Mode-K cells were lysed
in 13Laemmli buffer and 20 mg of protein was subjected to
electrophoresis (10% SDS-PAGE gels). Anti-p-RelA (Cell
Signaling, Danvers, USA), Anti-grp78 (Sigma Aldrich, Munich,
Germany), anti-p-eiF2a(Cell Signaling), anti-cleaved caspase 3
(Cell Signaling), TNF (Cell Signaling), ferritin (FTH1; Cell
Signaling) and anti-
b
-actin (MP Biomedicals, Illkirch, France)
were used to detect immunoreactive p-RelA, grp78, p-eiF2a,
cleaved caspase 3, ferritin and
b
-actin respectively, using an
enhanced chemiluminescence light-detecting kit (Amersham,
Arlington Heights, Illinois, USA).
Non-haem measurement
Livers and ileum were collected and 30 mg tissue was dissolved
in acid mix (6 M HCl, 20% v/v TCA). After incubation (658C,
20 h) and centrifugation (3000 rpm, 3 min), non-haem iron
content was measured photometrically using a commercial kit
following the manufacturers instructions (Feren-B, Bioanalytic,
Umrich/Freiburg, Germany).
Haematocrit and haemoglobin determination
Blood was drawn from each animal from the vena cava inferior
at the killing time point of 18 weeks. Haematocrit and haemo-
globin concentrations were measured using the micro-
haematocrit method (No. 749311; Brand, Wertheim, Germany;
Haematocrit-centrifuge 2104; Hettlich, Tuttlingen, Germany)
and the cyanmethaemoglobin method (reagent: Bioanalytic
4001; Umrich, Freiburg, Germany; photometer: UV-DK-20;
Beckmann, Munich, Germany), respectively. Mean corpuscular
haemoglobin concentration (MCHC) values were calculated by
using the formula (haemoglobin/haematocrit) 3100.
Isolation and stimulation of CD4
+
T cells from spleen
See supplemental material.
Ileal explant cultivation
See supplemental material.
Isolation of mesenteric CD8a
b
T cells
See supplemental material.
Epithelial cell and/or intraepithelial lymphocytes (IEL)
co-culture experiments
See supplemental material.
Cell-mediated cytotoxicity assay
For quantication of cell-mediated cytotoxicity, the Cellular
DNA Fragmentation ELISA (Roche, Mannheim, Germany) was
used. The assay was performed according to the manufacturer s
standard protocol.
ELISA analysis
TNF, interferon
g
(INF
g
) or Granzyme B protein concentrations
were determined in supernatant of ileal explant cultivation,
blood samples or medium from co-culture experiments at
different time points, by mouse-specic ELISA kits according to
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the manufacturers instructions (R&D Systems, Wiesbaden-
Nordenstadt, Germany).
Microbial analysis using 454 pyrosequencing
See supplemental material.
Statistical analysis
Statistical tests were performed using unpaired t-test or one-
way ANOVA followed by the HolmeSidak test. Differences
were considered signicant if values were <0.05(*) or <0.01(**),
<0.001(***).
RESULTS
Chronic ileitis is associated with induction of ER stress and
apoptosis in primary ileal IEC
Histological examination (score 0e12) of TNF
D
ARE/WT
mice
at the age of 18 weeks revealed severe ileitis in TNF
D
ARE/WT
mice (8.3060.91) in contrast to healthy WT mice (1.2060.29)
(gure 1A). H&E stained distal ileal segments from severely
inamed TNF
D
ARE/WT
mice show massive leucocyte inltration,
villus atrophy and crypt hyperplasia (gure 1B).
TNF
D
ARE/WT
mice represent a CD8
+
T cell-driven model
where disease progression is associated with a loss of CD8aa
+
and an increase in CD8a
b
+
IEL as major T effector cell pheno-
type.
9 13
We conrmed loss of CD8aa
+
and an increase of
CD8a
b
+
IEL in TNF
D
ARE/WT
compared to WT mice (gure 1C).
Since ER stress in IEC has been discussed in the pathogenesis
of IBD, we isolated primary ileal IEC to evaluate ER stress
responses in TNF
D
ARE/WT
mice. Five animals from each of the
two groups were pooled and 25 mg of total protein was used for
western blot analysis. As shown in gure 1D, we detected
increased expression levels of the major ER chaperone grp-78 and
phosphorylated eukaryotic initiation factor 2a(p-eiF2a)in
TNF
ΔARE/WT
mice compared to WT mice. In addition to ER
stress response mechanisms, we detected phosphorylated/acti-
vated nuclear factor kappa B (NF-
k
B) subunit RelA (p-RelA) as
well as enhanced protein expression of apoptosis-related cleaved
caspase 3 (cc3) in severely inamed TNF
ΔARE/WT
mice.
Systemic iron repletion does not extenuate the protective effect
of luminal iron sulfate deprivation
We next sought to evaluate the role of luminal iron sulfate on
the development of experimental ileitis in TNF
ΔARE/WT
mice
independent from the depletion of systemic iron stores (liver and
spleen). Therefore, we fed WT (N¼5) or TNF
ΔARE/WT
(N¼8)
mice an iron sulfate free diet. In parallel we treated these
mice with weekly intraperitoneal injections of FeNTA (iron-
repleted) or NaCl (Ctrl injection). The third group of WT and
TNF
D
ARE/WT
mice received the iron sulfate containing diet in
combination with weekly applied NaCl Ctrl injections. Histo-
logical examination of the distal ileum revealed absence of
inammation in TNF
ΔARE/WT
mice receiving an iron sulfate free
diet combined with NaCl Ctrl injections as well as FeNTA-
injections (gure 2A). In contrast, TNF
ΔARE/WT
mice receiving
the iron sulfate diet combined with NaCl-Ctrl-injections were
severely inamed. WT groups showed no signs of inammation.
H&E staining of representative parafn-embedded distal ileal
segments from TNF
D
ARE/WT
mice receiving the iron sulfate free
diet combined with NaCl Ctrl injections as well as FeNTA
injections conrmed the markedly reduced tissue pathology
compared to severely inamed TNF
D
ARE/WT
mice on the iron
sulfate containing diet (gure 2C). As shown in gure 2B, iron
repletion of WT and TNF
D
ARE/WT
mice on the iron sulfate free
diet restored at least 50% of hepatic non-haem iron content
compared to level of WT and TNF
D
ARE/WT
mice fed with the
iron sulfate containing diet.
Haematocrit (Hc) levels of iron-repleted TNF
D
ARE/WT
mice
showed no signicant differences to control mice (supplemen-
tary gure 1A). In contrast, WT and TNF
D
ARE/WT
mice on an
iron sulfate free diet showed signicantly reduced hepatic non-
haem iron content associated with signicantly reduced Hc
levels compared to all other groups. There were no signicant
changes in haemoglobin levels between the treatment groups
(supplementary gure 1B). Table 1 shows the calculated MCHC
values of all groups, demonstrating no signicant differences
between the different treatments. These results suggested that
the iron sulfate free diet almost completely depleted hepatic iron
stores without shifting the mice to an anaemic state.
Luminal iron deprivation reduced ER stress and apoptosis in IEC
Inamed TNF
D
ARE/WT
mice showed enhanced ER stress
responses and apoptosis in primary IEC when compared to
healthy WT mice (gure 1D). To further elucidate the role of
luminal versus systemic iron to trigger ER stress responses and
pro-apoptotic signals in the intestinal epithelium, we performed
an additional experiment with TNF
ΔARE/WT
mice receiving an
iron sulfate free diet. Consistent with the experiment described
above, systemic iron repletion of TNF
D
ARE/WT
mice (1.6760.20)
maintained the protective effect as observed in TNF
D
ARE/WT
mice (1.5460.25) receiving the iron sulfate free diet and NaCl
Ctrl injections in contrast to severely inamed TNF
D
ARE/WT
mice (7.0060.63) on the iron sulfate containing diet and NaCl
Ctrl injections. Iron repletion of iron sulfate free fed mice
(158.61627.36 mg Fe/g liver) was completely effective to restore
Figure 1 TNF
D
ARE/WT
mice as model for human Crohn’s disease-like
ileits. Histopathological analysis of WT and TNF
D
ARE/WT
mice (A) with
representative H&E staining of ileal segments (B). Alterations in CD8aa
+
/
CD8a
b
+
IEL T cell numbers between WT and TNF
D
ARE/WT
mice (C). ER
stress-, inflammation and apoptosis-associated protein expression in
purified primary ileal epithelial cells from inflamed TNF
D
ARE/WT
mice
compared to healthy WT mice (D). ER, endoplasmic reticulum; IEL,
intraepithelial lymphocytes; TNF, tumour necrosis factor; WT, wild type.
Werner T, Wagner SJ, Martı
´
nez I, et al.Gut (2010). doi:10.1136/gut.2010.216929 3 of 9
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hepatic non-haem iron content compared to level of the iron
sulfate containing diet (115.55628.04 mg Fe/g liver). Western
blot analysis revealed a slight inhibition of RelA phosphoryla-
tion in primary IEC from non-inamed TNF
ΔARE/WT
mice
receiving an iron sulfate free diet as well as iron-repleted
TNF
ΔARE/WT
compared to inamed TNF
ΔARE/WT
mice (gure 3A)
receiving the iron sulfate containing diet and NaCl Ctrl injec-
tions. However, western blot analysis showed absence of ER
stress and apoptosis in primary ileal epithelial cells under
luminal iron deprivation (gure 3A, middle row). Interestingly,
systemic iron repletion did not affect ER stress levels in IEC, but
revealed slightly enhanced protein expression levels for cc3
compared to iron sulfate free fed TNF
ΔARE/WT
mice with NaCl
Crtl injections (gure 3A, right row). To further evaluate IEC
iron levels, we performed a ferritin western blot. Consistent
with the non-haem iron measurement in total ileal tissue
(supplementary gure 2), we detected enhanced levels of ferritin
in TNF
ΔARE/WT
mice fed with the iron sulfate containing diet
compared to mice on the iron sulfate free diet. Although the
systemic iron repletion led to enhanced expression levels of
ferritin compared to NaCl Crtl injected mice, the strongest
expression of ferritin in IEC was shown for TNF
ΔARE/WT
mice
receiving an iron sulfate containing diet (gure 3A).
We next cultivated distal ileal segments from TNF
ΔARE/WT
mice of the second injection experiment for 24 h to determine
TNF expression. Consistent with the degree of tissue pathology,
ELISA analysis revealed signicantly elevated levels of TNF in
the inamed group receiving the iron sulfate containing diet
and NaCl Crtl injections (74.10616.91 pg/ml) compared to
the group receiving the iron sulfate free diet and NaCl Ctrl
injections (31.58615.74 pg/ml) as well as FeNTA injections
(35.1666.01 pg/ml) (gure 3B). Blood samples were taken to
measure TNF serum concentrations at 18 weeks of age showing
no signicant differences between the three groups (gure 3C),
suggesting that absence of luminal iron sulfate mediates
protective effects on the ileal tissue rather than affecting
systemic inammatory responses.
Luminal iron sulfate deprivation and systemic iron repletion did
not influence phenotype and activity of IEL
To evaluate the role of luminal iron sulfate deprivation and
systemic iron repletion on IEL phenotype, we performed ve
colour surface staining and FACS analysis. Iron-repleted
TNF
ΔARE/WT
mice showed a marginal, but signicant, loss of
CD4
+
T cells when compared to TNF
ΔARE/WT
mice on the iron
sulfate containing or iron sulfate free diet (gure 4A) with NaCl
Ctrl injections. Feeding of an iron sulfate free diet as well as iron
repletion in TNF
D
ARE/WT
mice had no signicant effect on
Figure 2 Systemic iron repletion did
not adversely affect the protective effect
of the iron sulfate free diet. Histological
analysis of WT and TNF
D
ARE/WT
mice
fed with an iron sulfate containing diet
or iron sulfate free diet in combination
with weekly applied NaCl Ctrl injections
or FeNTA injections (A). Non-heme iron
content of liver (B). Representative H&E
staining of ileal segments (C). Ctrl,
control; FeNTA, Fe(NO
3
)
3
complexed
with nitrilotriacetic acid; TNF, tumour
necrosis factor; WT, wild type.
Table 1 Values of the mean corpuscular haemoglobin concentration
Treatment Wild type TNF
D
ARE/WT
FeSO
4
in diet, NaCl Ctrl injection 28.5661.29 29.2162.80
No FeSO
4
in diet, NaCl Ctrl injection 27.8061.87 27.9162.61
No FeSO
4
in diet, FeNTA injection 27.2961.33 26.9660.81
Ctrl, control; FeNTA, Fe(NO
3
)
3
complexed with nitrilotriacetic acid.
4 of 9 Werner T, Wagner SJ, Martı
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    Gut microbiota dysbiosis has been considered the essential element in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Antibiotics were administered orally to Dark Agouti (DA) rats early in their life with the aim of perturbing gut microbiota and investigating the effects of such intervention on the course of EAE. As a result, the diversity of the gut microbiota was reduced under the influence of antibiotics. Mainly, Firmicutes and Actinobacteria were replaced by Proteobacteria and Bacteroidetes, while decreased proportions of Clostridia and Bacilli classes were accompanied by an increase in Gamma-Proteobacteria in antibiotic-treated animals. Interestingly, a notable decrease in the Helicobacteraceae, Spirochaetaceae and Turicibacteriaceae was scored in antibiotic-treated groups. Also, levels of short chain fatty acids were reduced in the faeces of antibiotic-treated rats. Consequently, aggravation of EAE, paralleled with stronger immune response in lymph nodes draining the site of immunization, and increased inflammation within the CNS, were observed in antibiotic-treated DA rats. Thus, the alteration of gut microbiota leads to an escalation of CNS-directed autoimmunity in DA rats. The results of this study indicate that antibiotic use in early life may have subsequent unfavourable effects on the regulation of the immune system.
  • Article
    Full-text available
    Gut microbiota interacting with an intact mucosal surface are key to the maintenance of homeostasis and health. This review discusses the current state of knowledge of the biofilm mode of growth of these microbiota communities, and how in turn their disruptions may cause disease. Beyond alterations of relative microbial abundance and diversity, the aim of the review is to focus on the disruptions of the microbiota biofilm structure and function, the dispersion of commensal bacteria, and the mechanisms whereby these dispersed commensals may become pathobionts. Recent findings have linked iron acquisition to the expression of virulence factors in gut commensals that have become pathobionts. Causal studies are emerging, and mechanisms common to enteropathogen-induced disruptions, as well as those reported for Inflammatory Bowel Disease and colo-rectal cancer are used as examples to illustrate the great translational potential of such research. These new observations shed new light on our attempts to develop new therapies that are able to protect and restore gut microbiota homeostasis in the many disease conditions that have been linked to microbiota dysbiosis.
  • Article
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    Ferrous sulfate is the most commonly used drug for treatment of iron deficiency anemia, but it is badly absorbed and causes many unfavorable side effects. Nanotechnology is a way to decrease the side effects of drugs and to increase the drug bioavailability. So, this study is designed to investigate the effect of iron oxide nanoparticles in comparison to ferrous sulphate in the treatment of iron deficiency anemia in rats. Forty male albino rats were divided into two main groups: Control group (10 rats) and anemic group (30 rats) that received standard iron free basal diet for six weeks. Then the anemic group was subdivided into three groups (10 rats) in each group: anemic control group, ferrous sulfate group (received ferrous sulfate 0.4 mg/kg b.w/ 10 days) and iron oxide nanoparticles group (received iron oxide nanoparticles 0.4 mg/kg b.w/ 10 days) in the drinking water. Iron oxide nanoparticlescaused a significant increase in the level of red blood cells (8.80±0.05 106/μL), hemoglobin (18.46±0.33 g/dL), hematocrit (46.66±0.23 %), mean corpuscular volume (MCV) (53.02±0.3 FL), mean corpuscular hemoglobin concentration (MCHC) (39.56±0.6 %), ferritin (447.6±9.02 μg/L), transferrin saturation (138.0±1.5), total iron binding capacity (TIBC) (145.00±1.15 mg/dL) and serum iron (276.33±2.07 mg/dL). Moreover, it decreased serum malondialdehyde (MDA) (31.85±0.34 nmol/g) and C-reactive protein (CRP) (312.66±1.7 mg/L) when compared to ferrous sulfate group and anemia control groups. These results revealed that iron oxide nanoparticles proved as an effective drug for the treatment of iron deficiency anemia in rats. Keywords: Anemia, Blood picture, Iron nanoparticle, Oxidative stress, Inflammatory indicators, Rats.
  • Article
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    Iron plays a key role in many physiological processes; cells need a very exact quantity of iron. In patients with inflammatory bowel disease, anaemia is a unique example of multifactorial origins, frequently being the result of a combination of iron deficiency and anaemia of chronic disease. The main cause of iron deficiency is the activity of the disease. Therefore, the first aim should be to reach complete clinical remission. The iron supplementation route should be determined according to symptoms, severity of anaemia and taking into account comorbidities and individual risks. Oral iron can only be used in patients with mild anaemia, whose disease is inactive and who have not been previously intolerant to oral iron. Intravenous iron should be the first line treatment in patients with moderate-severe anaemia, in patients with active disease, in patients with poor tolerance to oral iron and when erythropoietin agents or a fast response is needed. Erythropoietin is used in a few patients with anaemia to overcome functional iron deficiency, and blood transfusion is being restricted to refractory cases or acute life-threatening situations.
  • Article
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    Inflammatory bowel disease (IBD) is associated with anaemia and oral iron replacement to correct this can be problematic, intensifying inflammation and tissue damage. The intestinal microbiota also plays a key role in the pathogenesis of IBD, and iron supplementation likely influences gut bacterial diversity in patients with IBD. Here, we assessed the impact of dietary iron, using chow diets containing either 100, 200 or 400 ppm, fed ad libitum to adult female C57BL/6 mice in the presence or absence of colitis induced using dextran sulfate sodium (DSS), on (i) clinical and histological severity of acute DSS-induced colitis, and (ii) faecal microbial diversity, as assessed by sequencing the V4 region of 16S rRNA. Increasing or decreasing dietary iron concentration from the standard 200 ppm exacerbated both clinical and histological severity of DSS-induced colitis. DSS-treated mice provided only half the standard levels of iron ad libitum (i.e. chow containing 100 ppm iron) lost more body weight than those receiving double the amount of standard iron (i.e. 400 ppm); p<0.01. Faecal calprotectin levels were significantly increased in the presence of colitis in those consuming 100 ppm iron at day 8 (5.94-fold) versus day
  • Preprint
    Patients taking oral iron supplementation often suffer from gastrointestinal side effects. We have previously shown that acute alterations in oral iron exacerbate dextran sodium sulphate (DSS) induced colitis and are associated with dysbiosis. As patients take iron supplementation for long periods, we asked whether this too would influence colitis and the microbiome. We assessed the impact of long-term changes in dietary iron, by feeding chow containing 100ppm, 200ppm and 400ppm (reflecting a deficient, normal or supplemented diet, respectively) for up to 9 weeks to female wild-type C57BL/6 (WT) mice in presence or absence of chronic colitis, or acute colitis induced after 8 weeks, induced by DSS. Assessment was made based on (i) clinical and histological severity of colitis, and (ii) faecal microbial diversity, as assessed by sequencing the V4 region of 16S rRNA. In mice with long term changes to their dietary iron, reduced iron intake (100ppm iron diet) was associated with increased weight loss and histology scoring in the acute colitis model. Chronic colitis was not influenced by altering dietary iron however there was a clear change in the faecal microbiome in the 100 and 400ppm iron DSS-treated groups and in controls consuming the 400ppm iron diet. Proteobacteria levels increased significantly at day-63 compared to baseline and Bacteroidetes levels decreased in the 400ppm iron DSS group at day-63 compared to baseline; mirroring our previously published work in acute colitis. Long term dietary iron alterations clearly affects gut microbiota signatures but do not appear to exacerbate chronic colitis. However, acute colitis is exacerbated by changes in dietary iron. More work is needed to understand the impact of iron supplementation of the pathologenesis of IBD and rise that possiblity that the change in the microbiome, in patients with colitis, is a consequence of the increase in luminal iron and not simply the presence of colitis.
  • Article
    Background and Aim The prevalence of ulcerative colitis (UC) has been increasing in Japan. Trace elements such as iron, zinc, magnesium and copper, can cause digestive symptoms where there is a deficiency or excess. We focused on the dietary intake of trace elements and their associations with UC development. Methods A multicenter, hospital‐based case‐control study was conducted in Japan. Cases were 127 newly diagnosed UC patients and 171 age‐ and sex‐matched hospital controls were recruited. We considered that UC patients had potentially changed their dietary habits due to disease symptoms. The dietary habits were investigated using a self‐administered diet history questionnaire to analyze the dietary intakes and frequencies at two points, the previous one month and one year before. Results In the assessment of dietary habits one year before, the highest intake of iron showed an increased odds ratio (OR) for UC on multivariate analysis (OR = 4.05, 95% confidence interval (CI), 1.46‐11.2, P < 0.01). The highest intake of zinc one year before showed a decreased OR for UC (OR = 0.39, 95% CI, 0.18‐0.85, P = 0.01). Intakes of magnesium and copper had no significant association with UC. Since most UC cases had experienced the first symptom of UC within the previous 11 months, these intakes at one year before represented an association with pre‐illness dietary habits. Conclusion A high intake of iron has some effect on the development of UC. In contrast, a high intake of zinc has a protective effect on the development of UC.
  • Article
    Since the renaissance of microbiome research in the past decade, much insight has accumulated in comprehending forces shaping the architecture and functionality of resident microorganisms in the human gut. Of the multiple host-endogenous and host-exogenous factors involved, diet emerges as a pivotal determinant of gut microbiota community structure and function. By introducing dietary signals into the nexus between the host and its microbiota, nutrition sustains homeostasis or contributes to disease susceptibility. Herein, we summarize major concepts related to the effect of dietary constituents on the gut microbiota, highlighting chief principles in the diet-microbiota crosstalk. We then discuss the health benefits and detrimental consequences that the interactions between dietary and microbial factors elicit in the host. Finally, we present the promises and challenges that arise when seeking to incorporate microbiome data in dietary planning and portray the anticipated revolution that the field of nutrition is facing upon adopting these novel concepts.
  • Article
    To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.
  • Article
    Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation whose cellular components are capable of oxidative respiratory bursts that may result in tissue injury. Mucosal biopsies were analyzed for protein carbonyl content (POPs), DNA oxidation products [8-hydroxy-2-deoxyguanosine (8-OHdG)], reactive oxygen intermediates (ROIs), trace metals (copper, zinc, and iron) and superoxide dismutase (Cu-Zn SOD). In Crohn's disease biopsies, there was an increase in ROIs, POPs, 8-OHdG, and iron, while decreased copper and Cu-Zn SOD activity were found in inflamed tissues compared to controls. For ulcerative colitis, there was an increase in ROIs, POPs, and iron in inflamed tissue compared to controls, while decreased zinc and copper were observed. An imbalance in the formation of reactive oxygen species and antioxidant micronutrients may be important in the pathogenesis and/or perpetuation of the tissue injury in IBD and may provide a rationale for therapeutic modulation with antioxidants.
  • Article
    Full-text available
    To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.
  • Article
    The intestinal epithelial cell (IEC) is increasingly recognized to play a prominent role as an important intermediary between the commensal microbiota and the intestinal immune system. Moreover, it is now recognized that intestinal inflammation in inflammatory bowel disease (IBD) may arise primarily from IEC dysfunction due to unresolved endoplasmic reticulum (ER) stress as a consequence of genetic disruption of X box binding protein-1 function. In addition to primary (genetic) abnormalities of the unfolded protein response, a variety of secondary (inflammation and environmental) factors are also likely to be important regulators of ER stress. ER stress pathways are also well known to regulate (and be regulated by) autophagy pathways. Therefore, the host's ability to manage ER stress is likely to be a major pathway in the pathogenesis of intestinal inflammation that arises primarily from the IEC. Herein we discuss ER stress in the IEC as both an originator and perpetuator of intestinal inflammation in IBD.
  • Article
    Environmental factors substantially contribute to the development of chronic intestinal inflammation in the genetically susceptible host. Nutritional components like iron may act as pro-oxidative mediators affecting inflammatory processes and cell stress mechanisms. To better characterize effects of dietary iron on epithelial cell responses under the pathological conditions of chronic intestinal inflammation, we characterized the protein expression profile (proteome) in primary intestinal epithelial cells (IEC) from iron-adequate and low-iron fed wild-type (WT) and TNFΔARE/WT mice. We performed all possible comparisons between the 4 groups according to genotype or diet. Histological analysis of iron-adequate fed TNFΔARE/WT mice (∼0.54 mg of iron/day) revealed severe ileal inflammation with a histopathology score of 8.3 ± 0.91 (score range from 0-12). Interestingly, low-iron fed mice (∼0.03 mg of iron/day) were almost completely protected from the development of inflammatory tissue destruction (histopathology score of 2.30 ± 0.73). In total, we identified 74 target proteins with significantly altered steady state expression levels in primary IEC using 2D-gel electrophoresis (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS). Interestingly, the overlap between the comparison of iron-adequate fed WT and TNFΔARE/WT mice (inflamed conditions) and the comparison between the iron-adequate and iron-low fed TNFΔARE/WT mice (absence of inflammation) revealed 4 contrarily regulated proteins including aconitase 2, catalase, intelectin 1 and fumarylacetoacetate hydrolase (FAH). These proteins are associated with energy homeostasis, host defense, oxidative and endoplasmic reticulum (ER) stress responses. In conclusion, the iron-low diet affected the epithelial cell proteome and inhibited the development of chronic intestinal inflammation, suggesting a critical role for nutritional factors in the pathogenesis of IBD.
  • Article
    The unfolded protein response as a consequence of endoplasmic reticulum (ER) stress has recently been implicated as a novel mechanism that may lead to inflammatory bowel disease (IBD). Impairment of proper ER stress resolution in highly secretory Paneth and, to a lesser extent, goblet cells within the epithelium can primarily lead to intestinal inflammation. An inability to manage ER stress may not only be a primary originator of intestinal inflammation as exemplified by genetic polymorphisms in XBP1 that are associated with IBD but also a perpetuator of inflammation when ER stress is induced secondarily to inflammatory mediators or microbial factors. Furthermore, ER stress pathways may interact with other processes that lead to IBD, notably autophagy.
  • Article
    Full-text available
    Here, we describe an N-ethyl-N-nitrosourea (ENU)-induced missense error in the membrane-bound transcription factor peptidase site 1 (S1P)-encoding gene (Mbtps1) that causes enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis. S1P cleaves and activates cAMP response element binding protein/ATF transcription factors, the sterol regulatory element-binding proteins (SREBPs), and other proteins of both endogenous and viral origin. Because S1P has a nonredundant function in the ATF6-dependent unfolded protein response (UPR), woodrat mice show diminished levels of major endoplasmic reticulum chaperones GRP78 (BiP) and GRP94 in the colon upon DSS administration. Experiments with bone marrow chimeric mice reveal a requirement for S1P in nonhematopoietic cells, without which a diminished UPR and colitis develop.
  • Article
    Inflammatory bowel disease (IBD) has been attributed to aberrant mucosal immunity to the intestinal microbiota. The transcription factor XBP1, a key component of the endoplasmic reticulum (ER) stress response, is required for development and maintenance of secretory cells and linked to JNK activation. We hypothesized that a stressful environmental milieu in a rapidly proliferating tissue might instigate a proinflammatory response. We report that Xbp1 deletion in intestinal epithelial cells (IECs) results in spontaneous enteritis and increased susceptibility to induced colitis secondary to both Paneth cell dysfunction and an epithelium that is overly reactive to inducers of IBD such as bacterial products (flagellin) and TNFalpha. An association of XBP1 variants with both forms of human IBD (Crohn's disease and ulcerative colitis) was identified and replicated (rs35873774; p value 1.6 x 10(-5)) with novel, private hypomorphic variants identified as susceptibility factors. Hence, intestinal inflammation can originate solely from XBP1 abnormalities in IECs, thus linking cell-specific ER stress to the induction of organ-specific inflammation.
  • Article
    Previous studies suggesting increased reactive oxygen metabolite (ROM) production in inflammatory bowel disease have been restricted to peripheral blood and isolated intestinal phagocytes. In the current study, chemiluminescence and the effect of various scavengers, enzymes, and enzyme inhibitors were used to show that ROMs account for the increased production of oxidants by colorectal mucosal biopsy specimens in inflammatory bowel disease. Luminol-amplified chemiluminescence was increased in active ulcerative colitis [macroscopic grade 1: 25 photons.mg-1.min.10(-3) (median), 8-47 (95% confidence intervals), n = 40; grade 2: 89, 65-156, n = 30; grade 3: 247, 133-562, n = 13] and Crohn's disease [mild: 9, 3-84, n = 6; severe: 105, 25-789 (range), n = 5] compared with normal-looking mucosa (ulcerative colitis: 0.8, 0.4-1.4, n = 22, P less than 0.01; Crohn's disease: 0.8, 0.1-2, n = 6, P less than 0.05) and controls (0.6, 0.04-1.4, n = 52, P less than 0.01). In ulcerative colitis, luminol chemiluminescence correlated with microscopic inflammation (Spearman's p = 0.74, P = 0.0001) and was decreased by sodium azide (-89%, P less than 0.05), taurine (-31%, P less than 0.05), catalase (-23%, P less than 0.05), and dimethyl sulfoxide (-29%, P less than 0.05). Superoxide dismutase and oxypurinol decreased lucigenin chemiluminescence in ulcerative colitis by -63% (P less than 0.05) and -27% (P less than 0.05), respectively. Luminol chemiluminescence correlated with lucigenin chemiluminescence (Spearman's rho = 0.72, P = 0.003). These results suggest that neutrophil-derived oxidants (superoxide, hydrogen peroxide, hydroxyl radical, and hypochlorite) are generated in colorectal mucosa in active inflammatory bowel disease and support the hypothesis that production of such metabolites by neutrophils is of major pathogenetic importance.