IL-21 Enhances the Development of

Colitis-Associated Colon Cancer: Possible

This information is current as
of April 24, 2019.
Involvement of Activation-Induced Cytidine
Deaminase Expression
Akemi Araki, Lianjin Jin, Hidetoshi Nara, Yuji Takeda,
Nobuhito Nemoto, Md Yeashin Gazi and Hironobu Asao
J Immunol published online 24 April 2019
http://www.jimmunol.org/content/early/2019/04/23/jimmun
ol.1800550

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Supplementary http://www.jimmunol.org/content/suppl/2019/04/23/jimmunol.180055
Material 0.DCSupplemental

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Published April 24, 2019, doi:10.4049/jimmunol.1800550
The Journal of Immunology

IL-21 Enhances the Development of Colitis-Associated Colon

Cancer: Possible Involvement of Activation-Induced Cytidine
Deaminase Expression

Akemi Araki,* Lianjin Jin,* Hidetoshi Nara,* Yuji Takeda,* Nobuhito Nemoto,*,†
Md Yeashin Gazi,* and Hironobu Asao*
Inflammatory bowel diseases are known to be the origin of colitis-associated colon cancer (CAC). We previously reported that
dextran sulfate sodium (DSS)–induced colitis is exacerbated in mouse-IL-21-isoform transgenic (Tg) mice. In this study, we
assessed the CAC development induced by azoxymethane (AOM) and DSS in our Tg mice. AOM-DSS–induced tumor develop-
ment was dramatically increased in the Tg mice compared with wild-type mice. IL-21 is known to enhance activation-induced
cytidine deaminase (AID) expression in B cells and induce Ab class switching. In contrast, the AID expression in cells other than
B cells initiates tumor development in many tissues. Therefore, we investigated whether IL-21 induces the AID expression in the

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large intestinal epithelial cells (IECs) during CAC development. AID gene and protein expression was increased in the IECs of
AOM-DSS– or DSS-treated Tg mice compared with those of wild-type mice. Furthermore, we confirmed IL-21 induced AID gene
expression in the purified IECs ex vivo. The present study also showed IL-21R gene expression in unstimulated wild-type mouse
IECs, and this gene expression was augmented by TNF-a stimulation. The IL-21R expression and IL-21–induced AID gene
activation were further confirmed in the Colon-38 cell line. Taken together, IL-21 may be involved in increasing the risk of
CAC by enhancing the AID expression in IECs. The Journal of Immunology, 2019, 202: 000–000.

C
hronic inflammation is a major background factor for
carcinogenesis in humans (1–5), and ∼15–20% of human
malignant tumors are associated with chronic inflam-
mation (1, 2). Some types of colon cancer also occur on a
chronic inflammation background, such as ulcerative colitis;
therefore, ulcerative colitis is a risk factor for colorectal cancer
(6–9).
The multifunctional cytokine IL-21 is produced from activated
helper T cells such as Th17 cells and is reported to promote Th17
cell differentiation and proliferation (10). An accumulation of
inflammatory Th17 cells is one of the causes of inflammatory
bowel diseases, like ulcerative colitis (11, 12). Thus, to inves-
tigate the in vivo function of IL-21 in inflammatory bowel dis-
ease, we previously established mouse-IL-21-isoform transgenic
(Tg) mice and subjected them to dextran sulfate sodium (DSS)–
induced colitis. IL-21 isoform is an alternative splice variant
form of IL-21 that we previously identified and reported to have
*Department of Immunology, Yamagata University Faculty of Medicine, Yamagata
990-9585, Japan; and †Department of Orthopedics, Yamagata University Faculty of
Medicine, Yamagata 990-9585, Japan
ORCIDs: 0000-0002-0823-1802 (Y.T.); 0000-0002-1051-4799 (H.A.).
Received for publication April 17, 2018. Accepted for publication March 26, 2019.
This work was supported in part by Grants-in-Aid for Scientific Research 26460568
and 26930004 from the Japan Society for the Promotion of Science and a grant from
the Seizankai Medical Welfare Group.
Address correspondence and reprint requests to Prof. Hironobu Asao, Department of
Immunology, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata
990-9585, Japan. E-mail address: asao-h@med.id.yamagata-u.ac.jp
The online version of this article contains supplemental material.
Abbreviations used in this article: AID, activation-induced cytidine deaminase;
AOM, azoxymethane; CAC, colitis-associated colon cancer; CV, coefficient of
variation; DSS, dextran sulfate sodium; IEC, intestinal epithelial cell; MFI, mean
fluorescence intensity; qRT-PCR, quantitative real-time RT-PCR; Tg, mouse-IL-
21-isoform transgenic.
Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50
a similar function as original IL-21 (13, 14). The DSS-induced
colitis in the Tg mouse was much more severe and accompanied
by higher IL-17A production, compared with that in wild-type
mice (15). These findings suggested that IL-21 augmented the
Th17 cell development and exacerbated the colitis.
In this study, to expand on our previous findings, we in-
duced colitis-associated colon cancer (CAC) in our Tg mice
with a combination of DSS and azoxymethane (AOM), a potent
carcinogen (16). We found that the frequency and size of the
AOM-DSS–induced colon tumors in the Tg mice were sig-
nificantly higher and larger than in wild-type mice. Taken
together with previous reports that AOM-DSS–induced colon
tumor development is suppressed in IL-21–deficient mice (17, 18)
and that Th17 cells are associated with colon cancer devel-
opment (19), these results strongly suggest that IL-21 pro-
motes inflammation-induced colon carcinogenesis by increasing
the Th17 cells.
Follicular helper T cells also produce IL-21 to activate and
differentiate B cells. One of the important functions of IL-21 is to
induce class-switch recombination in activated B cells through
activation-induced cytidine deaminase (AID) expression (20–22).
AID is expressed only in activated B cells under physiological
conditions (23–25); however, it was also reported that AID-induced
gene mutation is involved in the development of human lympho-
cytic malignant tumor cells (26, 27). Furthermore, AID is expressed
in inflamed organs, and such expressed AID is reported to promote
carcinogenesis by causing genetic mutations and chromosomal
abnormalities (28, 29). In addition, AID-overexpressing trans-
genic mice develop epithelial tumors, such as liver cancer, lung
cancer, and gastric cancer, as well as malignant lymphoma (30,
31). Uncontrolled AID expression is also reported in human
colon cancer with ulcerative colitis (32, 33), stomach cancer
with Helicobacter pylori infection (34–36), liver cancer with
hepatitis C viral infection (37, 38), and oral squamous cell
carcinoma (39).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800550
2 IL-21 ENHANCES CAC DEVELOPMENT
In this study, we showed that IL-21 directly activated AID gene
expression in purified mouse intestinal epithelial cells (IECs)
through the IL-21R. These results suggest that IL-21 induces
AID expression via the IL-21R on IECs, leading to inflammatory
carcinogenesis.
Materials and Methods
Mice
BALB/c-background Tg mice were established previously (15). Mice
were maintained in a 12-h light/dark cycle under specific pathogen–free
conditions and had ad libitum access to a standard diet and water until
reaching the desired age (6–8 wk). All of the experiments with mice were
performed in accordance with the guidelines of the Laboratory Animal
Center of the Yamagata University Faculty of Medicine and were approved
by the Animal Experiment Committee of the Yamagata University Faculty
of Medicine.
Preparation of IECs
The large IECs of mice were collected as described previously, with some
modifications (17). Briefly, the large intestine was opened longitudi-
nally, thoroughly washed in ice-cold PBS, and cut into 1-cm segments.
The segments were placed in a 50-ml conical tube containing 15 ml of
PBS with 5 mM EDTA. The tube was placed horizontally on an orbital
shaker and shaken at 65 rpm for 30 min at 37˚C. The cell suspension
was passed through a mesh filter (100-mm pore size) to remove tissue
debris. This procedure was repeated twice. The cell suspension was
then centrifuged, and the cells were resuspended in RPMI 1640 medium
(Life Technologies) including 10% FCS. The collected cells were then
negatively purified using PE-CD45 (BioLegend) and BD IMag anti-PE
particles (BD Biosciences) according to the manufacturer’s instructions.
IECs were further purified from the CD45-negative cells by 25/45/75%
discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient centri-
fugation at 1000 3 g for 5 min at 4˚C. The IECs were collected from the
upper interphase of the Percoll gradient.
Cell lines and cell cultures
The murine colon cancer cell lines Colon-38 and Colon-26 were cultured in
RPMI 1640 medium containing 10% FCS and 100 mg/ml kanamycin.
The murine IEC line MIE was cultured in DMEM containing 10% FCS,
100 U/ml penicillin, and 10 mg/ml streptomycin. MIE cells were passaged
using a sucrose-EDTA buffer (0.45 M sucrose, 0.36% EDTA, 0.1% BSA in
PBS [pH 7.5]) as described (40).
Induction of colitis and CAC
For the DSS-induced colitis experiments, 4% (w/v) DSS (m.w. 5000; Wako
Pure Chemical Industries, Osaka, Japan) was administered in the drinking
water to 6- to 8-wk-old Tg mice for 13 d (15).
To induce CAC, 12 mg/kg AOM (Wako) dissolved in PBS was injected
i.p. into 6- to 8-wk-old wild-type and Tg mice on day 0. A week after the
AOM injection, the mice were given 2% DSS in the drinking water for a
week. After that, the mice were maintained for 20 wk with regular water
until the end of the experiment (Fig. 1A).
For the TNF-a inhibition in vivo, 13.5 mg/kg etanercept (Pfizer, Tokyo,
Japan) was injected s.c. into 6- to 8-wk-old wild-type mice every other day
for 15 d. At day 13 and 15, 4.5 mg/kg etanercept was injected. From 11 d
after etanercept injection, the mice were given 2% DSS in the drinking
water for 6 d (Supplemental Fig. 2A).
Histopathological analysis of AOM-DSS–induced colon cancer
The middle sections of colon tissue samples were excised, fixed with 10%
neutral buffered formalin, and embedded in paraffin. Round tissue sections
were thinly sliced and stained with H&E.
RNA isolation and quantitative real-time RT-PCR
Total RNA was prepared and amplified by RT-PCR, as described previ-
ously (13). For quantitative real-time RT-PCR (qRT-PCR) amplification,
THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) was used,
according to the manufacturer’s instructions. Thermocycling was performed
with a Rotor-Gene 6000 analyzer (Qiagen, Venlo, the Netherlands). The
primers used in this study are shown in Table I.
Flow cytometry analysis
To analyze cell-surface Ags, cells were stained with fluorescent dye–
conjugated mAbs. Pacific Blue–CD45, allophycocyanin-IL21R, and
allophycocyanin-CD132 (gc) were purchased from BioLegend. To
measure phosphorylated signal-transducing molecules, the cells were fixed
with Fixation Buffer (BioLegend) and permeabilized with 90% methanol
for 30 min on ice. The cells were then washed with wash buffer (PBS
containing 3% FCS and 0.02% NaN3), incubated with fluorescent dye–
conjugated mAbs on ice for 60 min, and then washed with wash buffer.
Alexa488-STAT3 (pY705), Alexa488-STAT1 (pY701), and Alexa647-
ERK1/2 (pT202/pY204) were purchased from BD Biosciences. The
solution 7-AAD was purchased from Affymetrix. To analyze AID ex-
pression, Zombie Aqua (BioLegend) negative cells were fixed with 4%
paraformaldehyde and 0.01% glutaraldehyde PBS and permeabilized
with 90% methanol for 30 min on ice. The cells were then washed with
wash buffer (PBS containing 3% FCS and 0.02% NaN3) and incubated
with anti-AICDA Ab (ab59361; abcam), followed by Alexa Fluor 488
goat-anti-rabbit IgG (A11008; Invitrogen). Normal rabbit IgG (X903;
DAKO) was used as control Ab. The stained cells were analyzed by
FACSCanto II (BD Biosciences).
Statistics
Results were expressed as means 6 SD. The statistical significance of
the data was evaluated by Student t test. In the case of AID expression
(Figs. 3F, 4E), comparison in the group was performed by Kruskal–
Wallis test with Steel test. Data analysis was performed using EZR
(Saitama Medical Center, Jichi Medical University, Saitama, Japan),
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which is a graphical interface for R (The R Foundation for Statistical
Computing, Vienna, Austria). It is a modified version of R commander
(41). A p value , 0.05 was regarded as significant.
Results
AOM-DSS–induced colon cancer is increased in Tg mice
The DSS colitis in Tg mice is much more severe than that in wild-
type mice (15). In this study, we induced CAC by administering
AOM in addition to DSS in our Tg and wild-type mice, as shown
in Fig. 1A. Induced CACs are indicated by arrows in Fig. 1B.
Significantly more tumors were observed in the Tg than in the
wild-type mice (Fig. 1C). The tumor load, which is the sum of
the tumor diameters, was also significantly increased in the Tg
mice (Fig. 1D). These increases in the Tg mice were similar in
both genders (Fig. 1C, 1D). Histologically, the tumors tended
to be larger in the Tg than in the wild-type mice (Fig. 1E). AOM-
DSS–induced CAC is repressed in IL-21–deficient mice (17, 18),
and Th17 inflammatory cells have an important role in CAC de-
velopment (19). Our results indicated that, like IL-21, IL-21 isoform
promotes the development of inflammation-induced carcinogenesis.
AID expression in the IECs is increased in Tg versus
wild-type mice
AID is expressed in many tumors, whereas IL-21 induces AID
expression physiologically in B cells. We hypothesized that the
AID gene expression in the IECs during the tumorigenesis may be
increased in Tg mice. First, we measured the AID gene expres-
sion in the colon tissues from DSS-treated mice. The AID gene
expression was significantly increased in the colon tissue of DSS-
treated Tg versus wild-type mice (Fig. 2A). Because colon tissue
includes activated B cells, which express AID physiologically, we
purified the IECs from colon tissue and analyzed the AID ex-
pression in them. The IECs from AOM-DSS–treated Tg mice
expressed higher levels of AID mRNA than those from wild-type
mice (Fig. 2B). We confirmed upregulation of AID protein in the
IECs of Tg mice by flow cytometry analysis (Fig. 2C). These
results indicated that IL-21 may enhance the AID expression in
IECs and that the induced AID might be involved in the AOM-
DSS–induced CAC development.
IL-21 directly induces AID expression in IECs through the
IL-21R
To examine whether the AID gene activation signal was directly
transduced to IECs or not, we first analyzed the IL-21R expression on
The Journal of Immunology 3

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FIGURE 1. AOM-DSS–induced colon cancer is increased in Tg mice. (A) Protocol for AOM-DSS–induced CAC. (B) Representative macroscopic
appearance of the colon of wild-type (Wt) and Tg mice. Arrows indicate tumors. (C) Number of tumors in Wt (open circles) and Tg mice (closed circles).
Left and right panels show results from male and female mice, respectively. Data indicate mean 6 SD (n = 14 for Wt and n = 10 for Tg [male mice], n = 15
per group [female mice]). (D) Tumor load, determined by summing all of the tumor diameters (mm) for a given animal, in Wt (open circles) and Tg mice
(closed circles). Left and right panels show results from male and female mice, respectively. Data indicate mean 6 SD (n = 6 per group [male mice], n = 7
per group [female mice]). (E) Representative H&E-stained sections of the colon tissue samples from Wt and Tg mice. Numbers 1–4 represent the indicated
parts of the colon. Scale bar, 1 mm. *p , 0.05, ***p , 0.001.

the IECs. Colon IECs from wild-type mice were isolated and purified
as described in the Materials and Methods, and 7-AAD–negative
and CD45-negative cells in the purified IECs were gated as IECs
(Fig. 3A). The IL-21R is a heterodimer consisting of IL-21R and gc
(42, 43). We found a low but significant expression of both subunits
(Fig. 3B, 3C). The mRNA expression was examined by RT-PCR and
qRT-PCR experiments. In the purified IECs, both mRNAs were
clearly detected in the RT-PCR experiment, even in the unsti-
mulated IECs (Fig. 3D). Next we examined whether the inflamed
condition affected the IL-21R expressions or not ex vivo. Purified
IEC from normal colon was treated with TNF-a, and the transcript
and protein of IL-21R were measured. Transcript of both receptors
tended to increase after TNF-a treatment (Fig. 3E). However, the
protein level of both receptors was not changed (Supplemental
Fig. 1A). So, we tried to suppress TNF-a function in vivo and
then measured the receptor expressions. We used TNF-a antago-
nist etanercept, which blocks TNF-a function in vivo (44, 45).
Etanercept treatment significantly reduced DSS-induced IL-21R
expression (Supplemental Fig. 2B). In contrast, gc expression did
not change. These results suggested that TNF-a functioned on the
4 IL-21 ENHANCES CAC DEVELOPMENT

Table I. Primers used in RT-PCR and qRT-PCR

Primers

RT-PCR and qRT-PCR

b-actin Forward: 59-TGACAGGATGCAGAAGGAGA-39
Reverse: 59-GCTGGAAGGTGGACAGTGAG-39
AID Forward: 59-CGTGGTGAAGAGGAGAGATAGTG-39
Reverse: 59-CAGTCTGAGATGTAGCGTAGGAA-39
gc Forward: 59-CGGAGCAACAGAGATCGAAG-39
Reverse: 59-TGAGAACTTCCACAGATTGGG-39
RT-PCR
IL-21R Forward: 59-ACAGCGGGAACTTCAAGAAATGGG-39
Reverse: 59-GTCACTGTGTCAATGGACACCAGA-39
qRT-PCR
IL-21R Forward: 59-CGGGAACTTCAAGAAATGGG-39
Reverse: 59-TCTTCTCCTTGGCTGGATAC-39

IL-21R protein expression in vivo, and certainly other inflamma- These results were quite similar to the results obtained with
tory cytokines may also be involved. Next, to confirm the AID primary IECs. Taken together, these findings suggested that
expression in the IECs in vivo (Fig. 2B, 2C), purified IECs from mouse IECs express the IL-21R and respond to ligand stim-
wild-type mice were stimulated ex vivo with IL-21 and/or TNF-a. ulation, especially under inflamed conditions.

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The AID mRNA level was significantly increased with IL-21 or
TNF-a, and the combination of both cytokines tended to increase
the level further (Fig. 3F). These results indicated that IL-21 di-
rectly activates the AID gene in IECs through its receptor.
IL-21 activates the AID gene in Colon-38 colon cancer cells
through the IL-21R
Next, we analyzed the IL-21R expression and IL-21–induced
AID gene expression in the Colon-38 colon cancer cell line
to confirm the results of the experiments in primary IECs. We
again found low but significant expression of both receptor
subunits (Fig. 4A, 4B). Both mRNA expressions were clearly
shown in the RT-PCR experiment and were significantly en-
hanced after TNF-a stimulation (Fig. 4C, 4D). TNF-a–induced
upregulation of IL-21R protein expression was confirmed
by flow cytometry experiments (Supplemental Fig. 1B). We also
detected the IL-21R expression in another mouse colon cancer
cell line, Colon-26, and in a mouse small IEC line, MIE
(Supplemental Fig. 3A–D). The AID mRNA level was signif-
icantly increased with IL-21 or TNF-a, and the combination of
both cytokines tended to further increase the level (Fig. 4E).
IL-21 induces signal transduction in colon epithelial cells
We further examined the signal transduction through the IL-21R in
the IECs and Colon-38 cells. As a positive control, IL-21 stimula-
tion clearly activated STAT3, STAT1, and ERK1/2, as shown by the
phosphorylation of these signal transducers (Fig. 5, mean fluorescence
intensity [MFI]) in splenocytes. We also monitored the decrease in
coefficient of variation (CV) as an indication of signaling op-
eration (Fig. 5) (46). In the Colon-38 cells and splenocytes,
STAT3, STAT1, and ERK1/2 were activated upon IL-21 stim-
ulation (Fig. 5, upper and lower panels). Whereas STAT3 acti-
vation was not clear in the IECs, STAT1 and ERK1/2 activation
were significantly increased, as indicated by the increased MFI and
decreased CV (Fig. 5, middle panels). The representative histograms
are shown in Supplemental Fig. 4.
Discussion
In this study, we demonstrated that IL-21 directly activated AID
gene in inflamed mouse colon epithelial cells through its receptor
and that IL-21 might lead to inflammatory carcinogenesis. Mouse
colon epithelial cells and mouse colon tumor cell lines were

FIGURE 2. AID gene expression in the colon or IECs is increased in Tg mice. (A) Wt and Tg mice were given 4% DSS for 13 d or water as a control.
Colon samples from Wt (white bars) and Tg mice (black bars) were analyzed for AID gene expression. Results were normalized to the expression of b-actin
mRNA. Data indicate mean 6 SD (n = 5 per group). (B and C) IECs were purified as described in Materials and Methods after only 10 d of CAC induction.
AID mRNA levels were normalized to the expression of b-actin mRNA (B), and AID protein levels were shown as MFI–fluorescence minus one (FMO)
(C). Data indicate mean 6 SD (n = 4 per group). *p , 0.05, **p , 0.01. ns, not significant.
The Journal of Immunology 5

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FIGURE 3. IL-21 directly induces AID gene expression in IECs through the IL-21R. (A) 7-AAD2 and CD452 cells were gated as IECs from purified
wild-type mouse IECs and analyzed for expression of the IL-21R. (B) Representative histogram data of the IL-21R and gc expression in IECs. (C) MFI of
IL-21R and gc on IECs (black columns). White columns indicate isotype control Ab staining (n = 6 per group). (D) Purified mouse IECs were cultured with
or without 25 ng/ml TNF-a for 15 h. Total RNA and cDNA were prepared as described in Materials and Methods. Representative RT-PCR gel electro-
phoresis data. Splenocytes were used as a positive control (pc). (E) The total RNA samples used in (D) were also analyzed for IL-21R gene expression by
qRT-PCR as described in Materials and Methods. Results were normalized to the expression of b-actin mRNA. Data indicate mean 6 SD (n = 4 for
IL-21R, and n = 6 for gc). (F) Purified IECs were stimulated with 25 ng/ml TNF-a, 1 nM IL-21, or both for 15 h and then analyzed for AID gene ex-
pression. Results were normalized to the expression of b-actin mRNA. Data indicate mean 6 SD (n = 3 for TNF-a and IL-21 stimulation, and n = 5 for the
others). *p , 0.05, **p , 0.01. nc, negative control; ns, not significant.

reported to not express the IL-21R (17), and IL-21 mainly acts on
immunological cells, such as T cells, B cells, NK cells, dendritic
cells, macrophages, and others. However, nonimmunological
cells like human colon epithelial cells, colon carcinoma cells,
gastric epithelial cells, and keratinocytes also express the
IL-21R (47–53). In this study, we clearly detected the IL-21R on
mouse IECs, Colon-38 and Colon-26 colon cancer cells, and
MIE small intestine epithelial cells by flow cytometry, RT-PCR,
and qRT-PCR analyses. Furthermore, the IL-21R expression
was enhanced by TNF-a treatment. TNF-a–induced enhance-
ment of IL-21R expression was very clear in Colon-38 cell but
not in IEC ex vivo (Supplemental Fig. 1). One reason for this
discrepancy may be the damage of separated IEC. So, we
tried to block the TNF-a signal in vivo, and etanercept
blocked IL-21R expression (Supplemental Fig. 2). Our re-
sults strongly suggest that the IL-21R is expressed on mouse
colon epithelial cells, especially under inflamed conditions,
at least with TNF-a.
STAT3 and IL-21 are important factors for the development of
colon cancer (54, 55). IL-21 was thought to promote the inflam-
matory status by inducing the production of IL-6, IL-17A, and
other factors and then by indirectly activated STAT3 (55, 56).
However, our study showed that it is also possible that IL-21
directly activates STAT3 in IECs and enhances AID expression,
6 IL-21 ENHANCES CAC DEVELOPMENT

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FIGURE 4. IL-21 activates the AID gene in Colon-38 colon cancer cells through the IL-21R. (A) Representative histogram data of the IL-21R and gc
expression in Colon-38 cells. (B) MFI of IL-21R and gc on Colon-38 cells (black columns). White columns indicate isotype control Ab staining (n = 6 per
group). (C) Colon-38 cells were cultured with or without 25 ng/ml TNF-a for 12 h. Representative RT-PCR gel electrophoresis data are shown. Splenocytes
were used as a positive control (pc). (D) The total RNA samples used in (C) were also analyzed for IL-21R gene expression by qRT-PCR. Results were
normalized to the expression of b-actin mRNA. Data indicate mean 6 SD (n = 4 per group). (E) Colon-38 cells were stimulated with 25 ng/ml TNF-a, 1
nM IL-21, or both for 12 or 24 h and then analyzed for AID gene expression. Results were normalized to the expression of b-actin mRNA. Data indicate
mean 6 SD (n = 5 in the nonstimulated and TNF-a–stimulated groups, n = 4 in the IL-21–stimulated and both-stimulated groups). *p , 0.05, **p , 0.01.
nc, negative control.

leading to CAC. In our ex vivo IEC experiment, the IL-21–
induced STAT3 activation was not significant, but a tendency of
STAT3 to be activated by IL-21 stimulation was observed. In con-
trast, IL-21–induced STAT1 and ERK1/2 activation were signifi-
cantly shown in the same cells. Despite ex vivo experiments that
showed AID gene activation (Fig. 3F), we could not detect
IL-21–induced AID protein expression in the same cells and also
in the Colon-38 cells (data not shown). AID expression was
reported to be posttranscriptionally downregulated by miRNA
(57). AID protein expression may require other signals in addition
to IL-21 and TNF-a ex vivo. Details of the signaling cascade for
the increase in AID gene expression and protein expression should
be elucidated in future studies.
We previously showed that DSS-induced colitis was severe in
BALB/c-background female Tg mice versus wild-type mice (15),
whereas there were no large differences between the BALB/
c-background male Tg or C57BL/6-background Tg mice and
their wild-type counterparts (A. Araki, unpublished observations).
In contrast, in this study, the AOM-DSS–induced CAC development
was increased in both male and female Tg mice and in both the
BALB/c and C57BL/6 backgrounds (data not shown). It takes only
2 wk for DSS-induced colitis to develop, whereas 20 wk is required
for the development of AOM-DSS–induced CAC. Although we do
not know the reason for this discrepancy, the longer experimental
period for the development of CAC compared with that for DSS
colitis may have caused the transgene to have a stronger effect
on the outcome.
IL-21 also has an antitumor effect and is currently being tested
for treating metastatic malignant melanoma and renal cell car-
cinoma in clinical trials (58, 59). In contrast, IL-21 may worsen
colitis and enhance CAC, and an IL-21–neutralizing Ab was
reported to shrink mouse CAC (17). Therefore, inhibiting the
IL-21 and IL-21R system may be a strategy for preventing and
treating CAC.
In this study, our findings suggested that IL-21 is directly in-
volved in the induction of AID in IECs, leading to CAC devel-
opment. Clinically, it is becoming clear that controlling chronic
inflammation can suppress carcinogenesis, probably by decreasing
The Journal of Immunology
FIGURE 5. IL-21 activates intracellular signaling molecules in IECs and Colon-38 cells. IECs, Colon-38 cells, and splenocytes were stimulated with or
without 1 nM IL-21 for 20 min and then analyzed for the phosphorylation of STAT3, STAT1, and ERK1/2 by flow cytometry, as described in Materials and
Methods. The MFI and CV values are shown (n = 5 per group). *p , 0.05, **p , 0.01. ns, not significant.
the abnormal induction of genes in epithelial cells that are
necessary for tumor formation. Elucidating the detailed mechanism
of IL-21–mediated AID induction, a molecular process linking
chronic inflammation to genetic abnormalities, will improve
our understanding of carcinogenesis.
Acknowledgments
We thank Dr. Hisashi Aso (Tohoku University) for providing the MIE and
Dr. Naoya Fujita (Cancer Chemotherapy Center, Japanese Foundation for
Cancer Research) for providing the Colon-38 and Colon-26.
Disclosures
The authors have no financial conflicts of interest.
References
1. Kuper, H., H. O. Adami, and D. Trichopoulos. 2000. Infections as a major
preventable cause of human cancer. J. Intern. Med. 248: 171–183.
2. Grivennikov, S. I., F. R. Greten, and M. Karin. 2010. Immunity, inflammation,
and cancer. Cell 140: 883–899.
3. Chiba, T., H. Marusawa, and T. Ushijima. 2012. Inflammation-associated cancer
development in digestive organs: mechanisms and roles for genetic and epige-
netic modulation. Gastroenterology 143: 550–563.
7
Downloaded from http://www.jimmunol.org/ by guest on April 24, 2019
4. Mantovani, A., P. Allavena, A. Sica, and F. Balkwill. 2008. Cancer-related in-
flammation. Nature 454: 436–444.
5. Coussens, L. M., and Z. Werb. 2002. Inflammation and cancer. Nature 420:
860–867.
6. Eaden, J. A., K. R. Abrams, and J. F. Mayberry. 2001. The risk of colorectal
cancer in ulcerative colitis: a meta-analysis. Gut 48: 526–535.
7. Gupta, R. B., N. Harpaz, S. Itzkowitz, S. Hossain, S. Matula, A. Kornbluth,
C. Bodian, and T. Ullman. 2007. Histologic inflammation is a risk factor for
progression to colorectal neoplasia in ulcerative colitis: a cohort study. Gastro-
enterology 133: 1099–1105; quiz 1340–1341.
8. Rutter, M., B. Saunders, K. Wilkinson, S. Rumbles, G. Schofield, M. Kamm,
C. Williams, A. Price, I. Talbot, and A. Forbes. 2004. Severity of inflammation
is a risk factor for colorectal neoplasia in ulcerative colitis. Gastroenterology
126: 451–459.
9. van Hogezand, R. A., R. F. Eichhorn, A. Choudry, R. A. Veenendaal, and
C. B. Lamers. 2002. Malignancies in inflammatory bowel disease: fact or fiction?
Scand. J. Gastroenterol. Suppl. 37: 48–53.
10. Korn, T., E. Bettelli, M. Oukka, and V. K. Kuchroo. 2009. IL-17 and Th17 cells.
Annu. Rev. Immunol. 27: 485–517.
11. Maynard, C. L., and C. T. Weaver. 2009. Intestinal effector T cells in health and
disease. Immunity 31: 389–400.
12. Strober, W., and I. J. Fuss. 2011. Proinflammatory cytokines in the pathogenesis
of inflammatory bowel diseases. Gastroenterology 140: 1756–1767.
13. Rahman, M., H. Nara, T. Onoda, A. Araki, J. Li, T. Hoshino, and H. Asao. 2007.
Cloning and characterization of an isoform of interleukin-21. FEBS Lett. 581:
4001–4009.
14. Nara, H., M. Rahman, A. Araki, L. Jin, Y. Takeda, and H. Asao. 2013. IL-21
isoform is a membrane-bound ligand and activates directly interacted cells.
Cytokine 61: 656–663.
8 IL-21 ENHANCES CAC DEVELOPMENT
15. Araki, A., H. Nara, M. Rahman, T. Onoda, J. Li, F. M. Juliana, L. Jin, K. Murata,
Y. Takeda, and H. Asao. 2013. Role of interleukin-21 isoform in dextran sulfate
sodium (DSS)-induced colitis. Cytokine 62: 262–271.
16. De Robertis, M., E. Massi, M. L. Poeta, S. Carotti, S. Morini, L. Cecchetelli,
E. Signori, and V. M. Fazio. 2011. The AOM/DSS murine model for the study
of colon carcinogenesis: from pathways to diagnosis and therapy studies.
J. Carcinog. 10: 9.
17. Stolfi, C., A. Rizzo, E. Franzè, A. Rotondi, M. C. Fantini, M. Sarra, R. Caruso,
I. Monteleone, P. Sileri, L. Franceschilli, et al. 2011. Involvement of interleukin-
21 in the regulation of colitis-associated colon cancer. J. Exp. Med. 208: 2279–
2290.
18. Jauch, D., M. Martin, G. Schiechl, R. Kesselring, H. J. Schlitt, E. K. Geissler, and
S. Fichtner-Feigl. 2011. Interleukin 21 controls tumour growth and tumour immu-
nosurveillance in colitis-associated tumorigenesis in mice. Gut 60: 1678–1686.
19. De Simone, V., F. Pallone, G. Monteleone, and C. Stolfi. 2013. Role of TH17
cytokines in the control of colorectal cancer. OncoImmunology 2: e26617.
20. Konforte, D., N. Simard, and C. J. Paige. 2009. IL-21: an executor of B cell fate.
J. Immunol. 182: 1781–1787.
21. Ettinger, R., G. P. Sims, A. M. Fairhurst, R. Robbins, Y. S. da Silva, R. Spolski,
W. J. Leonard, and P. E. Lipsky. 2005. IL-21 induces differentiation of human
naive and memory B cells into antibody-secreting plasma cells. J. Immunol. 175:
7867–7879.
22. Kobayashi, S., N. Haruo, K. Sugane, H. D. Ochs, and K. Agematsu. 2009.
Interleukin-21 stimulates B-cell immunoglobulin E synthesis in human beings
concomitantly with activation-induced cytidine deaminase expression and dif-
ferentiation into plasma cells. Hum. Immunol. 70: 35–40.
23. Muramatsu, M., V. S. Sankaranand, S. Anant, M. Sugai, K. Kinoshita,
N. O. Davidson, and T. Honjo. 1999. Specific expression of activation-induced
cytidine deaminase (AID), a novel member of the RNA-editing deaminase
family in germinal center B cells. J. Biol. Chem. 274: 18470–18476.
24. Cascalho, M. 2004. Advantages and disadvantages of cytidine deamination.
J. Immunol. 172: 6513–6518.
25. Muramatsu, M., H. Nagaoka, R. Shinkura, N. A. Begum, and T. Honjo. 2007.
Discovery of activation-induced cytidine deaminase, the engraver of antibody
memory. Adv. Immunol. 94: 1–36.
26. Kinoshita, K., and T. Nonaka. 2006. The dark side of activation-induced cytidine
deaminase: relationship with leukemia and beyond. Int. J. Hematol. 83: 201–207.
27. Okazaki, I. M., A. Kotani, and T. Honjo. 2007. Role of AID in tumorigenesis.
Adv. Immunol. 94: 245–273.
28. Marusawa, H., A. Takai, and T. Chiba. 2011. Role of activation-induced cytidine
deaminase in inflammation-associated cancer development. Adv. Immunol. 111:
109–141.
29. Chiba, T., and H. Marusawa. 2009. A novel mechanism for inflammation-
associated carcinogenesis; an important role of activation-induced cytidine de-
aminase (AID) in mutation induction. J. Mol. Med. (Berl.) 87: 1023–1027.
30. Okazaki, I. M., H. Hiai, N. Kakazu, S. Yamada, M. Muramatsu, K. Kinoshita,
and T. Honjo. 2003. Constitutive expression of AID leads to tumorigenesis.
J. Exp. Med. 197: 1173–1181.
31. Morisawa, T., H. Marusawa, Y. Ueda, A. Iwai, I. M. Okazaki, T. Honjo, and
T. Chiba. 2008. Organ-specific profiles of genetic changes in cancers caused by
activation-induced cytidine deaminase expression. Int. J. Cancer 123: 2735–2740.
32. Endo, Y., H. Marusawa, T. Kou, H. Nakase, S. Fujii, T. Fujimori, K. Kinoshita,
T. Honjo, and T. Chiba. 2008. Activation-induced cytidine deaminase links be-
tween inflammation and the development of colitis-associated colorectal cancers.
Gastroenterology 135: 889–898, 898.e1–e3.
33. Gushima, M., M. Hirahashi, T. Matsumoto, K. Fujita, K. Ohuchida, Y. Oda,
T. Yao, M. Iida, and M. Tsuneyoshi. 2011. Expression of activation-induced
cytidine deaminase in ulcerative colitis-associated carcinogenesis. Histopathol-
ogy 59: 460–469.
34. Marusawa, H., and T. Chiba. 2010. Helicobacter pylori-induced activation-
induced cytidine deaminase expression and carcinogenesis. Curr. Opin. Immu-
nol. 22: 442–447.
35. Nagata, N., J. Akiyama, H. Marusawa, T. Shimbo, Y. Liu, T. Igari,
R. Nakashima, H. Watanabe, N. Uemura, and T. Chiba. 2014. Enhanced
expression of activation-induced cytidine deaminase in human gastric mucosa
infected by Helicobacter pylori and its decrease following eradication.
J. Gastroenterol. 49: 427–435.
36. Matsumoto, Y., H. Marusawa, K. Kinoshita, Y. Endo, T. Kou, T. Morisawa,
T. Azuma, I. M. Okazaki, T. Honjo, and T. Chiba. 2007. Helicobacter pylori
infection triggers aberrant expression of activation-induced cytidine deaminase
in gastric epithelium. Nat. Med. 13: 470–476.
37. Kou, T., H. Marusawa, K. Kinoshita, Y. Endo, I. M. Okazaki, Y. Ueda,
Y. Kodama, H. Haga, I. Ikai, and T. Chiba. 2007. Expression of activation-
induced cytidine deaminase in human hepatocytes during hepatocarcino-
genesis. Int. J. Cancer 120: 469–476.
38. Endo, Y., H. Marusawa, K. Kinoshita, T. Morisawa, T. Sakurai, I. M. Okazaki,
K. Watashi, K. Shimotohno, T. Honjo, and T. Chiba. 2007. Expression of
activation-induced cytidine deaminase in human hepatocytes via NF-kappaB
signaling. Oncogene 26: 5587–5595.
39. Nakanishi, Y., S. Kondo, N. Wakisaka, A. Tsuji, K. Endo, S. Murono, M. Ito,
K. Kitamura, M. Muramatsu, and T. Yoshizaki. 2013. Role of activation-
induced cytidine deaminase in the development of oral squamous cell car-
cinoma. [Published erratum appears in 2013 PLoS One. 8.] PLoS One 8:
e62066.
40. Kanaya, T., K. Miyazawa, I. Takakura, W. Itani, K. Watanabe, S. Ohwada,
H. Kitazawa, M. T. Rose, H. R. McConochie, H. Okano, et al. 2008. Differen-
tiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage.
Am. J. Physiol. Gastrointest. Liver Physiol. 295: G273–G284.
41. Kanda, Y. 2013. Investigation of the freely available easy-to-use software ‘EZR’
for medical statistics. Bone Marrow Transplant. 48: 452–458.
42. Asao, H., C. Okuyama, S. Kumaki, N. Ishii, S. Tsuchiya, D. Foster, and
K. Sugamura. 2001. Cutting edge: the common gamma-chain is an indispensable
subunit of the IL-21 receptor complex. J. Immunol. 167: 1–5.
43. Habib, T., S. Senadheera, K. Weinberg, and K. Kaushansky. 2002. The common
gamma chain (gamma c) is a required signaling component of the IL-21 receptor
and supports IL-21-induced cell proliferation via JAK3. Biochemistry 41: 8725–
8731.
44. Popivanova, B. K., K. Kitamura, Y. Wu, T. Kondo, T. Kagaya, S. Kaneko,
M. Oshima, C. Fujii, and N. Mukaida. 2008. Blocking TNF-alpha in mice re-
duces colorectal carcinogenesis associated with chronic colitis. J. Clin. Invest.
118: 560–570.
45. Wang, Q. T., Y. J. Wu, B. Huang, Y. K. Ma, S. S. Song, L. L. Zhang, J. Y. Chen,
Downloaded from http://www.jimmunol.org/ by guest on April 24, 2019
H. X. Wu, W. Y. Sun, and W. Wei. 2013. Etanercept attenuates collagen-induced
arthritis by modulating the association between BAFFR expression and the
production of splenic memory B cells. Pharmacol. Res. 68: 38–45.
46. Takeda, Y., H. Nara, and H. Asao. 2017. Analysis of signal transducers using
flow cytometry is useful for detection of contractive and fluctuating signals.
Yamagata Med. J. 35: 21–32.
47. Caruso, R., D. Fina, I. Peluso, C. Stolfi, M. C. Fantini, V. Gioia, F. Caprioli,
G. Del Vecchio Blanco, O. A. Paoluzi, T. T. Macdonald, et al. 2007. A functional
role for interleukin-21 in promoting the synthesis of the T-cell chemoattractant,
MIP-3a, by gut epithelial cells. Gastroenterology 132: 166–175.
48. Liu, Z., L. Yang, Y. Cui, X. Wang, C. Guo, Z. Huang, Q. Kan, Z. Liu, and Y. Liu.
2009. Il-21 enhances NK cell activation and cytolytic activity and induces Th17
cell differentiation in inflammatory bowel disease. Inflamm. Bowel Dis. 15:
1133–1144.
49. De Simone, V., G. Ronchetti, E. Franzè, A. Colantoni, A. Ortenzi, M. C. Fantini,
A. Rizzo, G. S. Sica, P. Sileri, P. Rossi, et al. 2015. Interleukin-21 sustains in-
flammatory signals that contribute to sporadic colon tumorigenesis. Oncotarget
6: 9908–9923.
50. Caruso, R., D. Fina, I. Peluso, M. C. Fantini, C. Tosti, G. Del Vecchio Blanco,
O. A. Paoluzi, F. Caprioli, F. Andrei, C. Stolfi, et al. 2007. IL-21 is highly
produced in Helicobacter pylori-infected gastric mucosa and promotes gelati-
nases synthesis. J. Immunol. 178: 5957–5965.
51. Caruso, R., E. Botti, M. Sarra, M. Esposito, C. Stolfi, L. Diluvio,
M. L. Giustizieri, V. Pacciani, A. Mazzotta, E. Campione, et al. 2009. In-
volvement of interleukin-21 in the epidermal hyperplasia of psoriasis. Nat. Med.
15: 1013–1015.
52. Leonard, W. J., R. Zeng, and R. Spolski. 2008. Interleukin 21: a cytokine/
cytokine receptor system that has come of age. J. Leukoc. Biol. 84: 348–356.
53. Spolski, R., and W. J. Leonard. 2008. Interleukin-21: basic biology and impli-
cations for cancer and autoimmunity. Annu. Rev. Immunol. 26: 57–79.
54. Grivennikov, S. I., and M. Karin. 2010. Dangerous liaisons: STAT3 and NF-
kappaB collaboration and crosstalk in cancer. Cytokine Growth Factor Rev. 21:
11–19.
55. Stolfi, C., F. Pallone, T. T. Macdonald, and G. Monteleone. 2012. Interleukin-21
in cancer immunotherapy: friend or foe? OncoImmunology 1: 351–354.
56. Terzić, J., S. Grivennikov, E. Karin, and M. Karin. 2010. Inflammation and colon
cancer. Gastroenterology 138: 2101–2114.e5.
57. Teng, G., P. Hakimpour, P. Landgraf, A. Rice, T. Tuschl, R. Casellas, and
F. N. Papavasiliou. 2008. MicroRNA-155 is a negative regulator of activation-
induced cytidine deaminase. Immunity 28: 621–629.
58. Skak, K., M. Kragh, D. Hausman, M. J. Smyth, and P. V. Sivakumar. 2008.
Interleukin 21: combination strategies for cancer therapy. [Published erratum
appears in 2008 Nat. Rev. Drug Discov. 7: 272.] Nat. Rev. Drug Discov. 7: 231–
240.
59. Chapuis, A. G., S. M. Lee, J. A. Thompson, I. M. Roberts, K. A. Margolin,
S. Bhatia, H. L. Sloan, I. Lai, F. Wagener, K. Shibuya, et al. 2016. Combined IL-
21-primed polyclonal CTL plus CTLA4 blockade controls refractory metastatic
melanoma in a patient. J. Exp. Med. 213: 1133–1139.