MucosalImmunology | VOLUME 4 NUMBER 2 | MARCH 2011
nature publishing group
In recent years, it has become evident that inflammatory bowel
disease (IBD) results from a deregulated and excessive immune
reactivity within the intestinal wall that is direct against luminal
antigens. 1,2 Nonetheless, mechanisms that contribute to
amplify and maintain the mucosal inflammation are not yet fully
understood. An intriguing hypothesis is that in IBD, the tissue-
damaging immune-inflammatory reaction is partly sustained
by a defective activity of counter-regulatory mechanisms. 3,4
Interleukin (IL)-25 (also known as IL-17E) is a recently
described member of the IL-17 cytokine gene family. 5 Distinct
from other IL-17 cytokine family members, IL-25 has been
shown to facilitate pathogenic T helper (Th)-2 cell responses 6,7
Indeed, studies in mice have shown that both transgenic expres-
sion of IL-25 and systemic administration of recombinant IL-25
increase the production of IL-4, IL-5, and IL-13, cause epithelial
cell hyperplasia, and facilitate the recruitment of inflammatory
cells into inflamed tissues. 6,8 – 10 In contrast, IL-25-deficient mice
infected with the parasitic helminthes Nippostrongylus brasiliensis
and Trichuris muris are unable to sustain Th2 cell responses and
fail to expel the parasites from the gut. 11,12
More recent studies have shown that IL-25 can regulate the
outcome of other immune responses. For example, studies
in murine models of autoimmunity have shown that IL-25
can negatively regulate the development and / or amplification
of Th17-mediated pathology and suppress the production of
bacteria-driven IL-23 production in the gut. 13,14 In line with
this, we have recently shown that CD14 + cells isolated from the
inflamed gut of patients with Crohn ’ s disease (CD) or ulcera-
tive colitis (UC), the major forms of human IBD, respond to
IL-25 by downregulating the synthesis of multiple inflamma-
tory cytokines. 15 Consistently, in vivo in mice, administration of
IL-25 prevented and cured experimental colitides. 15,16
Interestingly, analysis of IL-25 expression in the human gut
revealed that IL-25 is mostly made by subepithelial macro-
phages and that patients with IBD produce significantly less
IL-25 compared with controls. 15 It is thus conceivable that in
IBD, the defective IL-25 production can contribute to sustain
and / or amplify the pathogenic response. However, the mole-
cular mechanisms that account for IL-25 downregulation during
intestinal inflammation are not known. Hence, in this study, we
analyzed factors that regulate IL-25 in the human gut.
Interleukin-25 production is differently regulated
by TNF- ? and TGF- ? 1 in the human gut
D Fina 1 , E Franz è 1 , L Rovedatti 2 , GR Corazza 2 , L Biancone 1 , PP Sileri 3 , G Sica 3 , TT MacDonald 4 ,
F Pallone 1 , A Di Sabatino 2 and G Monteleone 1
An altered balance between effector and regulatory factors is supposed to sustain the tissue-damaging immune
response in inflammatory bowel disease (IBD). We have recently shown that in IBD, there is a defective synthesis of the
counter-regulatory cytokine, interleukin (IL)-25. In this study we investigated factors that control IL-25 production in the
gut. IBD patients produced less IL-25 when compared with normal controls. Stimulation of normal intestinal explants
with tumor necrosis factor- α (TNF- α ), but not interferon- γ (IFN- γ ) or IL-21, reduced IL-25 synthesis. Consistently, IL-25
production was enhanced by anti-TNF- ? both in vitro and in vivo . Upregulation of IL-25 was also seen in normal colonic
explants stimulated with transforming growth factor- β 1 (TGF- β 1). As in IBD, TGF- β 1 activity is abrogated by Smad7, we
next assessed whether inhibition of Smad7 with an antisense oligonucleotide enhanced IL-25 expression. Knockdown
of Smad7 was accompanied by an increase in IL-25 production. Data show that IL-25 production is differently regulated
by TNF- α and TGF- β 1 in the human gut.
1 Department of Internal Medicine, University “ Tor Vergata ” of Rome , Rome , Italy . 2 Department of Internal Medicine, Fondazione IRCCS Policlinico, San Matteo, Universit à
di Pavia , Pavia , Italy . 3 Department of Surgery, University “ Tor Vergata ” of Rome , Rome , Italy . 4 Institute of Cell and Molecular Science, Bart ’ s and the London School of
Medicine and Dentistry , London , UK . Correspondence: G Monteleone ( Gi.Monteleone@Med.uniroma2.it )
Received 9 July 2010; accepted 9 September 2010; published online 13 October 2010. doi: 10.1038/mi.2010.68
VOLUME 4 NUMBER 2 | MARCH 2011 | www.nature.com/mi
IL-25 is downregulated in IBD but not celiac disease
Initially, we sought to confirm that in IBD there is a defective
IL-25 production. To this end, we measured IL-25 quantita-
tively in total proteins extracted from whole mucosal samples
by enzyme-linked immunosorbent assay. A diminished expres-
sion of IL-25 was found in CD and UC samples when compared
with controls ( Figure 1a ). IL-25 was also analyzed in biopsy
samples taken from uninvolved and involved mucosal areas of
further eight IBD patients. IL-25 protein expression was signi-
ficantly diminished in the inflamed tissue when compared with
uninflamed tissue ( Figure 1b ). Total extracts prepared from
ileal and colonic biopsies of CD patients showed no signi-
ficant difference in terms of IL-25 production (not shown).
Moreover, there was no difference in IL-25 production between
IBD patients taking no drugs and those who were receiving
mesalazine or mesalazine + steroids ( Figure 1c ).
To assess whether IL-25 expression is downregulated in
other chronic intestinal inflammatory diseases, we next meas-
ured IL-25 in duodenal biopsies of patients with active celiac
disease and normal controls. A more pronounced expression of
IL-25 was seen in celiac disease patients when compared with
controls ( Figure 2a ). Moreover, analysis of IL-25 in biopsies
taken from three patients before and after 1 year of gluten-free
diet revealed that induction of remission was associated with a
reduced synthesis of IL-25 ( Figure 2b ), thus suggesting that in
celiac disease, gluten-induced immune response sustains IL-25
production. This hypothesis is supported by the demonstration
that treatment of duodenal biopsies of inactive celiac disease
patients with peptic-tryptic digest of gliadin enhanced IL-25
production ( Figure 2c ).
TNF- α negatively regulates IL-25 production in the gut
Next, we determined which factors control IL-25 production in
the gut. To this end, we stimulated colonic biopsies taken from
normal controls with various inflammatory cytokines that are
produced in excess in IBD tissue. 17 – 19 Tumor necrosis factor- ?
(TNF- ? ), but not interferon- ? (IFN- ? ) or IL-21, significantly
reduced IL-25 synthesis ( Figure 3 ). To confirm these data, we
cultured biopsies taken from IBD patients with infliximab or
control IgG. Treatment of IBD explants with infliximab led
to a significant increase in IL-25 production ( Figure 4a ). In
contrast, incubation of IBD samples with neutralizing IFN- ?
or IL-21 antibody did not alter IL-25 production (not shown).
Subsequently, IL-25 protein expression was evaluated in colonic
biopsies taken from six IBD patients before and after a successful
treatment with infliximab. Induction of clinical and endoscopic
remission by infliximab was accompanied by enhanced syn-
thesis of IL-25 ( Figure 4b ). Taken together, these data suggest
that TNF- ? is a negative regulator of IL-25 in the human gut.
To confirm that TNF- ? is overexpressed in IBD, biopsies taken
from the same IBD patients and controls, who were investigated
for IL-25 expression, were analyzed for the content of TNF- ?
RNA transcripts by real-time PCR. As expected, a more pro-
nounced TNF- ? – ? -actin RNA relative expression was seen in
CD (140 ± 48 arbitrary units) and UC (51.13 ± 15.68 arbitrary
units) in comparison with normal controls (11.78 ± 7.34
arbitrary units) ( P < 0.01).
TGF- β 1 enhances IL-25 production
IL-25-expressing cells are enriched in the normal colonic
mucosa. 15 However, factors / mechanisms that induce and sustain
Figure 1 Interleukin (IL)-25 protein synthesis is diminished in
the inflamed gut of patients with inflammatory bowel disease (IBD).
( a ) Quantitative analysis of IL-25 protein expression in mucosal samples
taken from 18 healthy controls (HC), 23 patients with ulcerative colitis
(UC), and 15 patients with Crohn ’ s disease (CD) as measured by
enzyme-linked immunosorbent assay (ELISA). Data are expressed as
mean ± s.d.; * P < 0.001. ( b ) Total proteins extracted from IBD mucosal
biopsies taken from involved and uninvolved areas of seven patients with
UC and two patients with CD were measured for IL-25 protein content by
ELISA. Data are expressed as mean ± s.d.; * P < 0.004. ( c ) IL-25 protein
was quantified in total proteins extracted from inflamed colonic biopsies
of five IBD patients receiving no therapy (w / o therapy) and seven IBD
patients treated with mesalazine (5-ASA) or mesalazine + steroids by
ELISA. Data are expressed as mean ± s.d. NS, not significant.
MucosalImmunology | VOLUME 4 NUMBER 2 | MARCH 2011
IL-25 in the human gut remain unknown. Therefore, the
next series of experiments were conducted to evaluate whether
IL-25 expression is regulated by transforming growth factor- ? 1
(TGF- ? 1), a cytokine that is highly produced in the normal
human gut, where it contributes to maintain mucosal homeo-
stasis. 20,21 Stimulation of normal colonic biopsies with TGF- ? 1
significantly enhanced IL-25 production ( Figure 5a ).
In IBD, there is a defective TGF- ? 1 activity because of high
levels of Smad7, an intracellular protein that inhibits TGF- ? 1-
driven intracellular signaling. 3,4,22 Indeed, analysis of Smad7
protein expression in our samples by western blotting confirmed
that Smad7 protein was increased in IBD patients in comparison
with normal controls. In particular, patients with CD and UC
had a median of Smad7 – ? -actin ratio of 0.75 and 0.56 densito-
metry arbitrary units, respectively (range 0.4 – 0.98 and 0.26 – 0.7,
respectively) that was significantly higher than that found in
normal controls (median 0.12; range 0.001 – 0.29; P < 0.002).
Therefore, we next determined whether in IBD, the diminished
production of IL-25 was partly related to Smad7. To address this
issue, we incubated IBD mucosal explants with Smad7 sense
or antisense oligonucleotides, and then analyzed IL-25 protein
by enzyme-linked immunosorbent assay. Treatment of IBD
samples with Smad7 antisense oligonucleotide led to a signi-
ficant upregulation of IL-25 synthesis ( Figure 5b ). The effec-
tive reduction of Smad7 protein expression in whole mucosal
biopsies incubated with Smad7 antisense oligonucleotides was
confirmed by western blotting (not shown).
Initially considered as a factor that expands Th2 cell responses, 6,7
IL-25 has been recently shown to regulate additional immune
pathways. For example, it is known that IL-25 inhibits the syn-
thesis of cytokines associated with Th1 and Th17 immunity
and that, in vivo in mice, administration of IL-25 prevents and
cures experimental colitides. 13 – 16 IL-25 is constitutively pro-
duced in the human intestine, but its expression is markedly
reduced in IBD. 15 Interestingly, a recent study showed that
IL-25 is produced by endothelial cells in the lumbar spinal cord
of normal mice and that IL-25-producing cells are reduced
after the development of experimental autoimmune encephalo-
myelitis, a murine model of multiple sclerosis. 13 IL-25 adminis-
tration ameliorated experimental autoimmune encephalomyelitis
induced by passive transfer of myelin oligodendrocyte glyco-
protein-reactive CD4 + T cells in recipient mice. 13 A decrease
of IL-25 expression in brain capillary endothelial cells was also
seen in patients with active multiple sclerosis lesions. 23 Taken
together, these findings support the anti-inflammatory action
Figure 2 Interleukin (IL)-25 production is increased in active celiac
disease. ( a ) Mucosal samples were collected from the duodenum of
13 healthy controls (HC) and 10 patients with active celiac disease and
measured for IL-25 protein content by enzyme-linked immunosorbent
assay (ELISA). Data are expressed as mean ± s.d.; * P = 0.003. ( b ) IL-25
was measured in total proteins extracted from duodenal biopsies
taken from 3 celiac disease patients before (active) and after (inactive)
1 year of gluten-free diet. ( c ) IL-25 protein synthesis in duodenal
biopsies taken from inactive celiac disease patients and cultured in
the presence or absence (medium) of peptic-tryptic digest (PT) of
gliadin for 24 h.
Figure 3 Tumor necrosis factor- ? (TNF- ? ) reduces interleukin (IL)-25
production in normal intestinal biopsies. IL-25 was measured in total
proteins extracted from normal colonic explants cultured with or without
(unstimulated (unst)) TNF- ? , interferon- ? (IFN- ? ), or IL-21 for 24 h. Data
are expressed as mean ± s.d. of six experiments; * P = 0.035; NS, not
VOLUME 4 NUMBER 2 | MARCH 2011 | www.nature.com/mi
of IL-25, and suggest that defects in IL-25 production can contri-
bute to sustain inflammatory responses in various organs.
In this study we analyzed factors that regulate IL-25 in the
human gut. Initially, we confirmed that IL-25 production is
downregulated in IBD tissue, by measuring the cytokine in whole
mucosal extracts of IBD patients and controls. Interestingly, in
IBD, IL-25 expression was reduced in the involved areas when
compared with uninvolved tissues, whereas the expression of
the cytokine in samples taken from uninflamed mucosa did not
differ from that seen in the colon of controls ( Figure 1a, b ).
Moreover, IL-25 production did not differ between IBD patients
taking no therapy and those who were receiving anti-inflam-
matory drugs. These observations, together with our previous
follow-up studies showing that medically induced remission
enhances IL-25 production, 15 indicate that downregulation of
IL-25 in IBD is not caused by current therapy.
Another important finding of this work is that IL-25 is highly
expressed in the inflamed gut of patients with celiac disease.
Although further experimentation is necessary to ascertain how
IL-25 is expressed in other gastrointestinal inflammatory condi-
tions, the above data would seem to suggest that the defective
production of IL-25 seen in IBD is not an epiphenomenon of
the ongoing inflammation, but it might rather rely on factors /
mechanisms that act selectively in IBD tissue. One such factor
could be TNF- ? , because it is highly produced in IBD but not
in celiac disease mucosa. 17,24,25 Indeed, stimulation of normal
colonic biopsies with TNF- ? reduced IL-25 expression, whereas
anti-TNF- ? upregulated IL-25 synthesis in IBD mucosa both
in vivo and in vitro . These data fit with the study by Sonobe
et al. 23 showing that TNF- ? inhibited IL-25 RNA expression in
the brain capillary endothelial cell line, MBEC4. In the same
work, Sonobe et al. 23 also documented a negative effect of
IFN- ? on IL-25 mRNA expression in MBEC4 cells. In contrast,
we were not able to see any significant change in IL-25 expression
Figure 4 Anti-tumor necrosis factor- ? (TNF- ? ) treatment enhances
interleukin (IL)-25 production in inflammatory bowel disease (IBD) tissue.
( a ) Mucosal samples were collected from the inflamed colon of three
patients with Crohn ’ s disease (CD) and four patients with ulcerative
colitis (UC) and cultured with infliximab or control IgG for 24 h. Total
proteins were then extracted and analyzed for IL-25 content by enzyme-
linked immunosorbent assay (ELISA). Each point represents the value
of IL-25 protein in mucosal samples taken from a single patient and
treated with control IgG or infliximab. Horizontal bars indicate the median;
* P = 0.015. ( b ) Mucosal samples were collected from five CD patients
and one UC patient immediately before and after a successful treatment
with infliximab and used to extract total proteins. IL-25 was analyzed
by ELISA. Each point represents the value of IL-25 protein in mucosal
samples taken from a single patient before and after infliximab therapy.
Horizontal bars indicate the median; * P = 0.030.
Figure 5 IL-25 is positively regulated by TGF- ? 1 signalling. ( a )
Transforming growth factor- ? 1 (TGF- ? 1) enhances interleukin (IL)-25
production in normal colonic explants. Mucosal colonic samples were
cultured in the presence or absence (unstimulated (unst)) of TGF- ? 1 for
24 h. Total proteins were then extracted and analyzed for IL-25 content
by enzyme-linked immunosorbent assay (ELISA). Data are expressed as
mean ± s.d. of four experiments; * P = 0.03. ( b ) Treatment of inflammatory
bowel disease (IBD) mucosal explants with Smad7 antisense
oligonucleotide upregulates IL-25 synthesis. Mucosal samples of active
IBD patients (4 Crohn ’ s disease (CD) and 1 ulcerative colitis (UC)) were
cultured with Smad7 sense (S) or antisense (AS) oligonucleotides for
48 h, and then used to extract total proteins. IL-25 was analyzed by
ELISA. Each point represents the value of IL-25 protein in mucosal
samples taken from a single patient and treated with Smad7 S or AS.
Horizontal bars indicate the median; * P = 0.03.
MucosalImmunology | VOLUME 4 NUMBER 2 | MARCH 2011
in normal mucosal explants following IFN- ? exposure. The reason
for this apparent discrepancy is not known, although it is con-
ceivable that IL-25 production is regulated in a cell- and tissue-
dependent manner. In line with this, it is the demonstration
that eosinophils, but not mast cells, from allergen-challenged
mice significantly upregulate IL-25 expression when exposed
to stem cell factor. 26 The demonstration that IL-25 is enhanced
in celiac disease but not CD mucosa, despite that active lesions
in both diseases are characterized by excessive synthesis of
IFN- ? , 18,27 supports this hypothesis further.
As IL-25 is constitutively produced by gut mucosal cells, and
evidence suggests that IL-25 contributes to dampen intestinal
inflammation, 15 we conducted further experimentation to iden-
tify molecules, which positively regulate IL-25 production. We
focused our work on TGF- ? 1, because this cytokine is highly
synthesized in the gut where it has a key role in promoting
counter-regulatory mechanisms. 20,21 Our ex vivo organ culture
studies indicate that in the normal gut, IL-25 production can
be enhanced by TGF- ? 1. This scenario is however somewhat
different from what we can image in IBD tissue, where the high
levels of Smad7 inhibit TGF- ? 1 signaling. 3,4 If so, one could
hypothesize that knockdown of Smad7 could eventually increase
IL-25 synthesis. Indeed, treatment of IBD biopsies with Smad7
antisense oligonucleotide led to a significant upregulation of
A caveat of these studies is that analysis of IL-25 expression
was however performed in biopsies and not in purified cell
types. We think it would be biologically relevant to examine
Smad7 and IL-25 in single mucosal cell populations in order to
ascertain if inhibition of Smad7 by antisense oligonucleotide
directly leads to IL-25 production. This should be performed
using subepithelial macrophages, because these are the cell
sources of IL-25 in the human gut. 15 Unfortunately, we were
not able to purify sufficient cells to carry out mechanistic stud-
ies. Hence, at the present time, we cannot exclude the possibil-
ity that the increased synthesis of IL-25 seen in IBD biopsies
treated with Smad7 antisense is secondary to a downregulation
of inflammatory pathways (e.g., TNF- ? ) rather than the result of
a direct effect of Smad7 on IL-25 expression. As TNF- ? does not
seem to be involved in the regulation of Smad7 in IBD, 3 – 4 we can
exclude the possibility that TNF- ? -mediated downregulation of
IL-25 expression that we observed in IBD tissue is secondary to
induction of Smad7.
In conclusion, our data confirm that IL-25 is downregulated
in IBD and suggest that such a defect can be perpetuated and
amplified by inflammatory pathways acting in IBD tissue.
Patients and samples . Mucosal biopsies were taken from the inflamed
areas of 15 patients with CD and 23 patients with UC. In 9 IBD (2 CD
and 7 UC) patients, biopsies were taken from both involved and unin-
volved mucosal areas. In 9 out of 15 CD patients, the primary site of
involvement was the terminal ileum, whereas the remaining patients
had a colonic disease. Two CD patients were receiving mesalazine,
9 patients were receiving steroids and mesalazine, and the remaining
were taking azathioprine. Among UC patients, 17 were on mesalazine,
whereas the remaining were taking mesalazine and steroids.
Additional mucosal samples were taken from intestinal resection speci-
mens of eight CD patients undergoing surgery because of a chronic active
disease poorly responsive to medical treatment. At the time of surgery,
four patients were receiving steroids and four patients were on antibiotics
and steroids. Moreover, biopsies was collected from five CD patients and
one UC patient before and 10 weeks after a successful therapeutic treat-
ment with anti-TNF- ? antibody (i.e . , infliximab). To evaluate whether
in IBD, IL-25 expression is influenced by therapy, colonic biopsies were
taken from 5 active patients (4 CD and 1 UC) taking no drug and 7
patients (3 CD and 4 UC) treated with mesalazine ( N = 4) or mesalazine
and steroids ( N = 3).
Healthy controls for the IBD study included colonic mucosal samples
taken from macroscopically and microscopically unaffected areas of 18
subjects undergoing colonoscopy for colorectal cancer screening ( N = 13)
or colon resection for colon cancer ( N = 5).
To evaluate how IL-25 is expressed in other chronic intestinal
inflammatory diseases, biopsies were taken from the duodenum of
ten patients with active celiac disease. The histopathological diagnosis
of celiac disease was based on typical mucosal lesions with crypt cell
hyperplasia and villous atrophy. All active celiac disease patients were
positive for antiendomysial and antitransglutaminase antibodies at the
time of diagnosis. Of these ten celiac disease patients, three underwent
a second endoscopy at 1 year after the initial diagnosis for the appear-
ance of dyspeptic symptoms. All these patients were on gluten-free
diet, in histological remission, and negative for antiendomysial and
antitransglutaminase antibodies at the time of the second endoscopy.
Controls for celiac disease study included duodenal biopsies taken
from ten individuals who underwent upper endoscopy for dyspeptic
symptoms, but had normal histology, no increase in inflammatory
cells, and were negative for antiendomysial and antitransglutaminase
antibodies. Informed consent was obtained from all patients and controls,
and the study protocol was approved by the local ethics committee.
Ex vivo organ cultures . All of the reagents were from Sigma-Aldrich
(Milan, Italy) unless specified. Freshly obtained mucosal samples were
cultured as described elsewhere. 3 Briefly, intestinal samples were placed
on iron grids with the mucosal face upward in the central well of an
organ culture dish in RPMI-1640 medium containing 10 % fetal bovine
serum, and supplemented with penicillin (100 U ml – 1 ) and streptomycin
(100 ? g ml – 1 ) (all from Lonza, Verviers, Belgium). Dishes were placed in
a tight container with 95 % O2 / 5 % CO2 at 37 ° C, at 1 bar. Control biopsies
were incubated in the presence or absence of TNF- ? (20 ng ml – 1 ), TGF- ? 1
(5 ng ml – 1 ), IL-21 (100 ng ml – 1 ; all from R & D Systems, Minneapolis,
MN), and IFN- ? (100 ng ml – 1 ; Peprotech, London, UK). Mucosal sam-
ples taken from the inflamed colon of four patients with CD and three
patients with UC were incubated with infliximab or control human IgG
(both at 50 ? g ml – 1 ). Additionally, samples taken from three patients with
CD were incubated in the presence or absence of a neutralizing IFN- ?
(5 ? g ml – 1 ; R & D Systems) or IL-21 (antibody 5 ? g ml – 1 ). 28 After 24 h of
culture, mucosal samples were snap frozen and stored at − 80 ° C until
To examine the effect of Smad7 inhibition on the expression of IL-25,
mucosal samples taken from four patients with CD and one patient with
UC were cultured in X-vivo, a serum-free culture medium, supplemented
with penicillin – streptomycin in the presence or absence of Smad7 anti-
sense or sense oligonucleotides (10 ? g ml – 1 ). These concentrations were
selected on the basis of our previous studies showing that at these con-
centrations Smad7 antisense oligonucleotide was effective in reducing
Smad7 expression in whole mucosal biopsies. 3 Both Smad7 antisense
and sense oligonucleotides were combined with Lipofectamine 2000
Reagent according to the manufacturer ’ s instructions (Invitrogen Italia,
San Giuliano Milanese, Italy). After 48 h, intestinal mucosal specimens
were snap frozen and stored at − 80 ° C until tested.
Duodenal biopsies were taken from three inactive celiac disease
patients and cultured with a peptic-tryptic digest of gliadin (1 mg ml – 1 )
for 24 h as previously described. 29
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VOLUME 4 NUMBER 2 | MARCH 2011 | www.nature.com/mi
Total protein extraction, IL-25 enzyme-linked immunosorbent assay,
and Smad7 western blotting . Intestinal mucosal samples were lysed on
ice in buffer containing 10 m M HEPES (pH 7.9), 10 m M KCl, 0.1 m M
EDTA, 0.2 m M EGTA, and 0.5 % Nonidet P40, supplemented with 1 m M
dithiothreitol, 10 mg ml – 1 aprotinin, 10 mg ml – 1 leupeptin, 1 m M phenyl-
methylsulfonyl fluoride, 1 m M Na 3 VO 4 , and 1 m M NaF. Lysates were
clarified by centrifugation at 12,000 g for 30 min at 4 ° C. Extracts were
analyzed for IL-25 content using a sensitive commercial enzyme-linked
immunosorbent assay kit (Peprotech) according to the manufacturer ’ s
instructions. Smad7 expression was analyzed by western blotting as
previously described 3 using a specific rabbit polyclonal antibody
(final dilution 1:500, Imgenex, San Diego, CA).
Real-time PCR . RNA was extracted using TRIzol reagent according to
the manufacturer ’ s instructions (Invitrogen). A constant amount of RNA
(1 ? g per sample) was retro-transcribed into complementary DNA, and
1 ? l of complementary DNA per sample was then amplified using the
following conditions: denaturation 1 min at 95 ° C, annealing 30 s at 62 ° C
for TNF- ? and at 60 ° C for ? -actin, followed by 30 s of extension at 72 ° C.
? -actin (FWD: 5-AAGATGACCCAGATCATGTTTGAGACC-3
and REV: 5-AGCCAGTCCAGACGCAGGAT-3) and TNF- ? (FWD:
5 ´ -AGGCGGTGCTTGTTCCTCAG-3 ´ and REV 5 ´ -GGCTACAGG
CTTGTCACTCG-3) expressions were evaluated using IQ SYBR Green
Supermix (Bio-Rad Laboratories, Milan, Italy). Real-time PCR was
performed using a CFX96 thermal cycler (Bio-Rad Laboratories). Gene
expression was calculated using the ? ? Ct algorithm.
Statistical analysis . Differences between groups were compared using
the paired t -test, the Mann – Whitney U -test, and one-way analysis of
This work received support from the ‘ Fondazione Umberto di Mario ’ ,
Rome, the Broad Medical Research Program Foundation (grant no.
IBD-0242), and Giuliani SpA, Milan, Italy.
G.M. has filed a patent entitled “ A treatment for inflammatory diseases ”
(patent Nr. 08154101.3).The remaining authors declared no conflict of
© 2011 Society for Mucosal Immunology
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