ORIGINAL ARTICLE
IL-9 and its receptor are predominantly involved
in the pathogenesis of UC
Nancy Nalleweg,
1
Mircea Teodor Chiriac,
1,2,3
Eva Podstawa,
1
Christian Lehmann,
4
Tilman T Rau,
5
Raja Atreya,
1
Ekaterina Krauss,
1
Gheorghe Hundorfean,
1
Stefan Fichtner-Feigl,
6
Arndt Hartmann,
5
Christoph Becker,
1
Jonas Mudter
1,7
▸ Additional material is
published online only. To view
please visit the journal online
(http://dx.doi.org/10.1136/
gutjnl-2013-305947).
For numbered affiliations see
end of article.
Correspondence to
Dr Jonas Mudter, Sana Clinic
Ostholstein, Hospitalstraße 22,
Eutin 23701, Germany; jonas.
mudter@sana.de
NN and MTC contributed
equally.
Received 26 August 2013
Revised 20 May 2014
Accepted 22 May 2014
Published Online First
23 June 2014
To cite: Nalleweg N,
Chiriac MT, Podstawa E,
et al. Gut 2015;64:
743–755.
ABSTRACT
Objective Several pathogenic roles attributed over the
past two decades to either T helper (Th)1 or Th2 cells
are increasingly becoming associated with interleukin
(IL)-17 and most recently IL-9 signalling. However, the
implication of IL-9 in IBD has not been addressed so far.
Design We investigated the expression of IL-9 and
IL-9R by using peripheral blood, biopsies and surgical
samples. We addressed the functional role of IL-9
signalling by analysis of downstream effector proteins.
Using Caco-2 cell monolayers we followed the effect of
IL-9 on wound healing.
Results IL-9 mRNA expression was significantly
increased in inflamed samples from patients with UC as
compared with controls. CD3
+
T cells were major
IL-9-expressing cells and some polymorphonuclear
leucocytes (PMN) also expressed IL-9. IL-9 was
co-localised with the key Th9 transcription factors
interferon regulatory factor 4 and PU.1. Systemically, IL-9
was abundantly produced by activated peripheral blood
lymphocytes, whereas its receptor was overexpressed on
gut resident and circulating PMN. IL-9 stimulation of the
latter induced IL-8 production in a dose-dependent
manner and rendered PMN resistant to apoptosis
suggesting a functional role for IL-9R signalling in the
propagation of gut inflammation. Furthermore, IL-9R
was overexpressed on gut epithelial cells and IL-9
induced STAT5 activation in these cells. Moreover, IL-9
inhibited the growth of Caco-2 epithelial cell monolayers
in wound healing experiments.
Conclusions Our results provide evidence that IL-9 is
predominantly involved in the pathogenesis of UC
suggesting that targeting IL-9 might become a
therapeutic option for patients with UC.
INTRODUCTION
UC, the most common form of IBD, is a chronic
condition in which the overreacting immune system
drives a sustained epithelial damage of the gut in
response to microbial and environmental factors in
a genetically predisposed individual.
12
Our under-
standing of IBD pathogenesis has profited from
major advances in experimental animal models of
the disease and sequence analysis of susceptibility
genes.
1–4
Based on patient data and the murine
oxazolone-induced colitis model, that drives a
Th2-like response, it has been proposed that epithe-
lial barrier damage in UC is driven by a T helper cell
(Th)2-like phenotype and interleukin (IL)-13 has
been associated with disease pathogenesis in UC.
56
The identification of the Th17 lineage of T
helper cells at the beginning of the century
7
has
challenged the Th1-Th2 paradigm proposed by
Significance of this study
What is already known on this subject?
▸ IBD is a chronic life-threatening condition
characterised by an overreacting immune
response in genetically susceptible individuals.
▸ Disease pathogenesis in UC, one of the two
major forms of IBD, has been attributed to an
altered Th2/Th17 response.
▸ Our previous work suggested a critical role for
interferon regulatory factor 4 (IRF4) in
controlling IL-17-dependent gut inflammation.
▸ IRF4 and PU.1 have been recently identified as
master transcription factors of the newly defined
Th9 cells.
What are the new findings?
▸ IL-9 was highly expressed in UC correlating with
disease activity; the main source of IL-9 were
CD3
+
T cells and multicolour
immunofluorescence stainings demonstrated the
co-localisation of IL-9 with the Th9 transcription
factors IRF4 and PU.1 but not with the cytokines
IL-4 or IL-10.
▸ Stimulated peripheral blood lymphocytes from
patients with UC produced significantly more IL-9
than control cells.
▸ Peripheral blood and lamina propria infiltrating
polymorphonuclear leucocytes expressed the
IL-9R; stimulation of blood polymorphonuclear
leucocytes with IL-9 resulted in a
dose-dependent production of IL-8 that (1)
could be blocked by anti-IL-9R antibodies and
(2) was accompanied by increased resistance of
polymorphonuclear leucocytes to apoptosis.
▸ IL-9 stimulation of freshly isolated
IL-9R-overexpressing epithelial cells led to STAT5
activation; anti-IL-9R blockade could prevent the
IL-9-induced growth delay of Caco-2 cell
monolayers in wound healing assays.
How might it impact on clinical practice in
the foreseeable future?
▸ IL-9 could represent a valuable tool in assessing
disease severity and its signalling pathways may
become novel therapeutic targets in UC.
Inflammatory bowel disease
Nalleweg N, et al. Gut 2015;64:743–755. doi:10.1136/gutjnl-2013-305947 743
group.bmj.com on January 24, 2017 - Published by http://gut.bmj.com/Downloaded from
Mosmann and Coffman in 1986
89
opening new avenues for
dissecting molecular patterns in cellular immunology. It was
soon demonstrated that Th17 cells were critically involved in
host defence and disease pathogenesis in tissues like the gut, the
lung and the brain.
710
At present, it is accepted that UC patho-
genesis is driven by a modified Th2 response mixed with a
Th17 cell-sustained inflammation.
10 11
Even more recently
however, new subsets of Th cells, for example, Th9
12
and
Th22,
13
have been defined but their roles in gut pathology
remained unaddressed.
IL-9 has been discovered as a potent growth factor for T cell
lines and mast cells and its role in allergic and asthmatic conditions
has been largely characterised over the past decade.
14 15
Initially
viewed as a Th2 cytokine, and thereafter associated with Th17
responses, IL-9 has been recently proposed to be the defining cyto-
kine for a new lineage of CD4 T cells, the Th9 cells, which add-
itionally produce high levels of IL-10 but none of the Th1-specific,
Th2-specific and Th17-specificcytokines.
16 17
The signalling of
the novel Th9 cells has been linked to the transcriptional pro-
grammes of STAT6,
16–18
interferon regulatory factor (IRF) 4
19
and PU.1.
20
Nevertheless, our previous studies demonstrated a
critical role for IRF4 in the pathogenesis of IBD.
21 22
In the present study, we investigated the role of IL-9 and the
Th9 transcription factors IRF4 and PU.1 in UC. Our findings
suggest that IL-9 is predominantly involved in the pathogenesis
of the disease.
MATERIALS AND METHODS
The collection of huma n samples was approved by the local
Ethical Committee and t he Review Board of the University of
Erlangen-Nuremberg, and each patient gave written informed
consent (number 4032/KFO/2011, 4032/2009). Materials and
methods are available as online supplementary file 1.
RESULTS
IL-9 mRNA is significantly overexpressed in mucosal
biopsies of the inflamed gut in UC
We first assessed colonic biopsies from patients with UC and
controls by quantitative real-time PCR for IL-9 mRNA expres-
sion. In these studies, mucosal inflammation was classified
according to previously described criteria: inflammation score
(IS)0 (absent inflammation)—IS3 (severe inflammation).
23
It was
found that the level of IL-9 mRNA expression increased with
increasing IS in UC (figure 1A). In addition, the levels of other
proinflammatory cytokines (such as IL-17A and IL-6) were also
increased in severely inflamed UC mucosa. However, IL-9 levels
in the ileum and colon of patients with Crohn’s disease were
not significantly upregulated (figure 1A). Moreover, in contrast
to IL-9, mRNA levels for IL-4, IL-6, IL-10, IL-13, IL-17A,
IL-21, IFNγ, TGF-β and TNFα were detected in patients with
UC with lower inflammation and, albeit at lower levels, also in
controls (figure 1A). Highest correlations between IL-9 mRNA
levels and levels of IL-6 (R=0.49; p<0.001), IL-13 (R=0.47;
p<0.001) and IL-17A (R=0.47; p<0.001) were observed
(figure 1B). In addition, a correlation between the expression
levels of the inflammatory protein marker S100A8 and IL-9 was
noted (R=0.57; p<0.001; figure 1B).
CD3
+
T cells are key IL-9-expressing cells in the
inflamed gut of patients with UC
Next, we systematically focused on investigating mildly (ie, IS1)
as well as severely (ie, IS3) inflamed material along with control
samples. Paraffin sections of gut specimens from patients with
UC with mild and severe inflammation and non-inflammatory
controls were first stained with antibodies against IL-9
(figure 2A). Quantification of these data showed a significantly
increased number of IL-9-expressing cells in severe versus mild
inflammation as well as in both groups versus the control group
(figure 2B). Furthermore, two-colour immunofluorescence stain-
ings of paraffin sections revealed that patients with severe
inflammation had significantly increased numbers of CD3
+
IL-9-expressing cells compared with patients with mild inflam-
mation and the control group, respectively (figure 2C, D).
Although CD3
+
cells accounted for more than 75% of the
IL-9-expressing cells in the inflamed gut (figure 2E), some
IL-9-expressing cells were CD3
−
. To address the nature of these
cells, we stained gut sections with IS3 for IL-9 and myeloperoxi-
dase, a specific marker for polymorphonuclear leucocytes
(PMN) that often dominate the acute inflammatory infiltrate in
UC. Our results showed that myeloperoxidase positive PMN
can express IL-9 in UC (see online supplementary figure S1A).
Upregulation of the key Th9 transcription
factors IRF4 and PU.1 in UC
IL-9 expr ession by Th9 cells was pre viously r eported to be depend-
ent on the transcription factors IRF4
19
and PU.1.
20
We found that
mRNA expression levels of IRF4 and PU.1 generally increased
with increasing inflammation in UC (figure 3A). Moreo ver, patients
with severe disease expressed significantly more IRF4 and PU.1
compared with controls which showed lower expression. Within
individual cells IL-9 co-localised with each of the two key transcrip-
tion factors of the Th9 cells, namely IRF4 and PU.1, respectively
(figure 3B, C). The number of IRF4
+
IL-9-expressing cells and
PU.1
+
IL-9-expressing cells was significantly increased in patients
with severe (ie, IS3) versus mild (ie, IS1) disease as well as in both
groups versus contr ols (figur e 3B, C).
To functionally address the role of IRF4 for IL-9 production
in UC, we isolated peripheral blood lymphocytes of patients
with UC and cultured them under Th9 polarising conditions.
We found that compared with non-polarised cells which only
had marginal levels of IL-9 expression, as many as 6% of the
Th9 polarised cells expressed IL-9 in patients with UC
(figure 4A). Moreover, we observed that 25% of the polarised
CD3
+
IRF4
+
cells expressed IL-9 (figure 4A). We additionally
performed three-colour immunofluorescence stainings on IS3
gut biopsies and found that IL-9 and IRF4 were coexpressed in
CD3
+
cells (figure 4B), consistent with the idea that IRF4 acts
as a key transcription factor for the generation of IL-9 in the
inflamed gut of patients with UC. IL-9 has been previously
found to be coexpressed with IL-10 in murine CD3
+
T cells
16
17
or with IL-4 in murine invariable natural killer (NK)T cells,
24
respectively. To address the expression profile of these cytokines
in IL-9-expressing cells, we stained freshly isolated lamina
propria cells from inflamed UC tissue with antibodies to CD3,
Vα24-Jα18 (invariant NKT cells marker), IL-4, IL-9 and IL-10.
Our results demonstrated that IL-9 and IL-10 were produced by
distinct CD3
+
cells (figure 4C). Moreover, invariant NKT cells
did not produce IL-9 (figure 4C). Taken together these results
suggest that in patients with UC, IL-9 is mainly produced by
CD3
+
cells that do not coexpress IL-10 or IL-4.
Human IL-9-secreting peripheral blood lymphocytes express
the gut homing ma rkers α4 and β7 integrin
To investigate the potential of peripheral blood lymphocytes to
produce IL-9 along with other cytokines whose levels have been
found to be altered in UC (eg, IL-4, IL-10, IL-13, IL17A/F,
Inflammatory bowel disease
744 Nalleweg N, et al. Gut 2015;64:743–755. doi:10.1136/gutjnl-2013-305947
group.bmj.com on January 24, 2017 - Published by http://gut.bmj.com/Downloaded from
TGF-β), we isolated and stimulated blood lymphocytes from UC
or healthy controls for two days with anti-CD3/anti-CD28 anti-
bodies. Stimulated UC lymphocytes produced significantly more
IL-9 compared with unstimulated cells from the same patients
(figure 5A). Cells from non-inflammatory controls that were sti-
mulated under the same conditions produced significantly less
Figure 1 Interleukin (IL)-9 overexpression in mucosal biopsies from patients with UC. Patients were scored according to histopathological criteria
for inflammation activity in UC
23
as described in materials and methods. (A) Quantitative assessment of IL-4, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-21,
IFN-γ, TGF-β and TNF-α mRNA expression in sigmoid colon and rectum biopsies of patients with UC with no (IS0) (n=7), mild (IS1) (n=11; for IL-9,
n=16), moderate (IS2) (n=9; for IL-9, n=27) and severe (IS3) (n=16; for IL-9, n=25) disease and healthy controls (n=10) shown as fold induction
(left). Quantitative assessment of IL-9 mRNA expression in patients with UC and ileal (n=9) and colonic biopsies (n=6) of patients with Crohn’s
disease with moderate and severe inflammation and healthy controls (right). CD, Crohn’s disease. IS, inflammation score. (B) Correlation of IL-9
mRNA levels with levels for IL-6, IL-13, IL-17A, IL-4, IL-10, IL-21, IFN-γ, TGF-β, TNF-α and S100A8 mRNA in patients with UC with detectable IL-9
levels (n=13 for cytokines, n=18 for S100A8). Data are shown as mean values±SEM (*p<0.05, **p<0.01, ***p<0.001, n.s., not significant) using
GraphPad Prism 5.
Inflammatory bowel disease
Nalleweg N, et al. Gut 2015;64:743–755. doi:10.1136/gutjnl-2013-305947 745
group.bmj.com on January 24, 2017 - Published by http://gut.bmj.com/Downloaded from
IL-9 when compared with stimulated UC cells, whereas
unstimulated control cells did not produce detectable levels of
IL-9. Furthermore, we found that stimulated peripheral blood
lymphocytes from patients with UC having C reactive protein
levels <5 mg/L produced significantly less IL-9 when compared
with patients with UC with C reactive protein levels >5 mg/L
Figure 2 Interleukin (IL)-9 expression by CD3
+
T cells is increased in the gut of patients with UC with severe inflammation. (A) Immunofluorescence
staining for IL-9 of paraffin sections of human mucosal tissues from controls and patients with UC with mild (IS1) and severe (IS3) inflammation.
(B) Quantification of the IL-9-expressing cells in 7 high-power fields per patient (n=5 per group). (C) Immunofluorescence two-colour staining for IL-9 and
CD3 of paraffin sections of mucosal tissues from controls, patients with mild (ie, IS1) and severe inflamed (ie, IS3) UC (inset: higher magnification).
Representative cells coexpressing IL-9 and CD3 are marked with an arrowhead. Scale bars, all 50 mm. (D) Quantifica tion of the CD3
+
IL-9-expressing cells
in 7 high-power fields per patient (n=5 per group). (E) Percentage of the CD3
+
IL-9-expressing and CD3
−
IL-9-expressing cells in 7 high-power fields per
patient (n=5 per group). Data are shown as mean values±SEM (**p<0.01, ***p<0.001) using GraphPad Prism 5. IS, inflammation score.
Inflammatory bowel disease
746 Nalleweg N, et al. Gut 2015;64:743–755. doi:10.1136/gutjnl-2013-305947
group.bmj.com on January 24, 2017 - Published by http://gut.bmj.com/Downloaded from
(figure 5A). IL-13 levels increased the most between healthy
individuals and patients with UC, although IL-10 (5.8 times)
and TGF-β (4.6 times) levels were also considerably upregu-
lated. Moderate correlations were observed between IL-9 and
IL-13 (R=0.69; p<0.001) and between IL-9 and IL-10 levels
(R=0.52; p<0.01), respectively (figure 5B). The occurrence of
IL-9 producing CD3
+
cells in the gut and the peripheral blood
raised the question of whether peripheral IL-9-expressing T cells
might home to the gut in UC. To address this experimentally,
we analysed the expression of α4 and β7 integrins in Th9
polarised blood CD3
+
T cells. We found that 16% of the integ-
rin α4
+
and 30% of the β7
+
CD3
+
cells expressed IL-9 (figure
5C) suggesting that circulating IL-9-expressing cells might
become recruited into the inflamed gut by their surface expres-
sion of α4 and β7 integrins.
IL-9R overexpression is functionally relevant for
antiapoptotic signals in UC
Based on previous data indicating increased IL-9R expression on
PMN from patients with asthma,
25
we sought to investigate the
IL-9R expression profile on peripheral blood and gut-infiltrating
PMN in patients with UC. We could demonstrate the presence
of the IL-9R on cytospins by immunofluorescence (see online
supplementary figure S1B). Using flow cytometry, we found that
PMN from patients with UC expressed significantly more IL-9R
compared with controls (figure 6A). Furthermore, CD16
−
eosi-
nophils also expressed IL-9R, albeit at a lower level (figure 6A).
To study functional aspects of IL-9 signalling, PMN were iso-
lated from the peripheral blood and stimulated with various
doses of recombinant IL-9. We observed a dose-dependent IL-8
production (figure 6B). This effect could be inhibited by pre-
blocking IL-9R signalling in PMN from patients with UC
(figure 6B). Granulocyte macrophage colony stimulating factor
(GM-CSF)-stimulated PMN from patients with UC that served
as stimulation controls produced significantly more IL-8 com-
pared with control PMN (figures 6B). Traditionally, PMN were
considered to be short-lived terminally differentiated cells
whose life span can be substantially increased by proinflamma-
tory stimuli.
26
To investigate the ability of IL-9 to modulate
PMN survival, we incubated cells with different doses of IL-9.
Figure 3 In UC lesions, interleukin (IL)-9 is coexpressed with the Th9 transcription factors interferon regulatory factor 4 (IRF4) and PU.1.
(A) Determination of IRF4 and PU.1 mRNA expression in the sigmoid colon and rectum biopsies of patients with UC with no (IS0) (n=7), mild (IS1) (n=11),
moderate (IS2) (n=9) and severe (IS3) (n=16) inflammation and healthy controls (n=10), respectively. (B, C) Two-colour immunofluorescence staining of
para ffin sections from control (left) and severe inflamed (ie, IS3) (middle) mucosal tissues for IL-9 and IRF4, and IL-9 and PU.1. Representative cells
coexpressing IL-9 and IRF4 or PU.1 are indicated by arrowheads. Scale bars, all 50 mm (inset: higher magnification). Quantification of the IRF4
+
IL-9
+
and
PU.1
+
IL-9
+
cells in patients with UC and controls is provided (right). Cells were counted in 7 high-power fields per patient (n=5 per group). Data are
shown as mean values±SEM (*p<0.05, **p<0.01, ***p<0.001) using GraphPad Prism 5. IS, inflammation score.
Inflammatory bowel disease
Nalleweg N, et al. Gut 2015;64:743–755. doi:10.1136/gutjnl-2013-305947 747
group.bmj.com on January 24, 2017 - Published by http://gut.bmj.com/Downloaded from