Human inflammatory bowel diseases (IBD) including
Crohn diseases and ulcerative colitis are chronic
immune-mediated disorders whose pathogenesis and
etiology are largely unknown (1). A number of gene-
manipulated murine models of colitis have been devel-
oped. These include IL-10–deficient, T cell receptor-
α–deficient, Gαi2–deficient, Stat4-transgenic, and
T-bet-transgenic mice (2–5). Experimentally induced
models of colitis have also been established by recon-
stituting immunocompromised mice with CD4+
CD45RBhighT cells. All of these models are used in
attempts to elucidate the pathogenesis of IBD. As a
result, it has been proposed that an exaggerated bias
toward Th1 or Th2 polarization in the mucosal immune
system is a key factor in the pathogenesis of colitis (6, 7).
In IL-10–deficient mice, CD4+T cells residing in the
lamina propria region of intestinal tract preferentially
produced IFN-γ (8). Immunocompromised mice trans-
ferred with T cells from IL-10–deficient mice developed
colitis with skewed Th1 responses (9). Furthermore,
treatment with anti–IFN-γ Ab significantly delayed the
onset of the disease and reduced the severity of colitis in
young IL-10–deficient mice (8). Treatment of anti-IL-12
Ab prevented the development of the disease in young
IL-10–deficient, but not in aged IL-10–deficient, mice (7,
10). Thus, the involvement of Th1 cells has become clear
in the pathogenesis of early phase of colitis in IL-10–defi-
cient mice. IL-10 is a well-known regulatory cytokine,
which can provide positive and negative signals on a vari-
ety of immune cells including B cells, T cells, NK cells,
mast cells, and myeloid cells (11). Additionally, IL-10 has
recently been shown to play an important role in the dif-
ferentiation and function of regulatory T cells, which
possess a capacity to inhibit harmful immune disorders
including chronic colitis (12, 13). Although it is now
The Journal of Clinical Investigation|May 2003| Volume 111|Number 9
production of IL-12p40 causes
chronic enterocolitis in myeloid
cell-specific Stat3-deficient mice
Masaya Kobayashi,1Mi-Na Kweon,2Hirotaka Kuwata,1Robert D. Schreiber,3
Hiroshi Kiyono,2,4Kiyoshi Takeda,1,5and Shizuo Akira1,5
1Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
2Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
3Department of Pathology and Immunology, Center for Immunology, School of Medicine, Washington University,
St. Louis, Missouri, USA
4Division of Mucosal Immunology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
5Solution Oriented Research for Science and Technology, Japan Science and Technology Corp., Tokyo, Japan
Stat3 plays an essential role in IL-10 signaling pathways. A myeloid cell-specific deletion of Stat3
resulted in inflammatory cytokine production and development of chronic enterocolitis with
enhanced Th1 responses in mice. In this study, we analyzed the mechanism by which a Stat3 defi-
ciency in myeloid cells led to the induction of chronic enterocolitis in vivo. Even in the absence of
Stat1, which is essential for IFN-γ signaling pathways, Stat3 mutant mice developed chronic entero-
colitis. TNF-α/Stat3 double-mutant mice developed severe chronic enterocolitis with enhanced Th1
cell development. IL-12p40/Stat3 double-mutant mice, however, showed normal Th1 responses and
no inflammatory change in the colon. RAG2/Stat3 double-mutant mice did not develop enterocoli-
tis, either. These findings indicate that overproduction of IL-12p40, which induces potent Th1
responses, is essential for the development of chronic enterocolitis in Stat3 mutant mice. Further-
more, enterocolitis was significantly improved and IFN-γ production by T cells was reduced in
TLR4/Stat3 double-mutant mice, indicating that TLR4-mediated recognition of microbial compo-
nents triggers aberrant IL-12p40 production by myeloid cells, leading to the development of entero-
colitis. Thus, this study clearly established a sequential innate and acquired immune mechanism for
the development of Th1-dependent enterocolitis.
J. Clin. Invest.111:1297–1308 (2003). doi:10.1172/JCI200317085.
Received for publication October 8, 2002, and accepted in revised form
January 28, 2003.
Address correspondence to: Shizuo Akira, Department of Host
Defense, Research Institute for Microbial Diseases, Osaka
University, 3-1 Yamada-oka, Suita Osaka 565-0871, Japan.
Phone: 81-6-6879-8302; Fax: 81-6-6879-8305;
Conflict of interest: The authors have declared that no conflict of
Nonstandard abbreviations used: inflammatory bowel diseases
(IBD); phycoerythrin (PE); Toll-like receptor 4 (TLR4).
See the related Commentary beginning on page 1284.
clear that IL-10 plays an essential role in the negative reg-
ulation of inflammatory responses in the intestine, the
mechanism by which IL-10 exerts the anti-inflammato-
ry responses remains unclear.
The STAT family of transcription factors, consisting
of seven members, is involved in cytokine signaling.
The knockout of each member of the STAT family in
mice resulted in impaired responses to corresponding
cytokines, demonstrating that STAT proteins have
essential roles in cytokine-mediated biological respons-
es (14, 15). Unlike the knockout of all other STAT
family members, the knockout of Stat3 resulted in
early death in embryogenesis (16). Therefore, Stat3
has been deleted in a cell- or tissue-specific manner by
the Cre-loxP recombination system in order to exam-
ine biological and functional roles of the transcription
factor in vivo (17–19). For example, mice lacking Stat3
in myeloid cells such as macrophages, neutrophils,
and dendritic cells developed chronic enterocolitis
and showed exaggerated Th1 responses (20). The
immunological and histological analysis revealed very
similar disease development in the intestinal tract of
IL-10–deficient mice. Macrophages from the Stat3
mutant mice did not show any anti-inflammatory
responses mediated by IL-10 and produced increased
levels of inflammatory cytokines in response to bacte-
rial LPS, demonstrating that Stat3 is essential for the
IL-10–mediated signaling pathways in myeloid cells
(20). This study also indicated that myeloid cells,
including macrophages and dendritic cells, are major
targets for IL-10 exhibiting anti-inflammatory
responses in vivo, and the abnormal activation of
myeloid cells is involved in the pathogenesis of chron-
ic colitis in mice. It remains elusive, however, how the
abnormal activation of myeloid cells leads to the
development of colitis in vivo.
In this study, we examined the in vivo mechanism by
which activated myeloid cells in the absence of IL-10 sig-
naling induce chronic colitis using Stat3 mutant mice
with additional deleted alleles, such as Stat1, TNF-α,
IL-12p40, RAG2, and Toll-like receptor 4 (TLR4).
Mice. Stat3 mutant (LysMcre; Stat3flox/flox) mice on a mixed
129 × C57BL/6 genetic background (backcrossed to
C57BL/6 for three generations) were used in this study
(17, 20). Stat1-deficient mice on a C57BL/6 background
were mated with Stat3 mutant mice (21). TNF-α–defi-
cient and IL-12p40–deficient mice on a C57BL/6 back-
ground were kindly provided by K. Sekikawa and J.
Magram, respectively (22, 23). RAG2-deficient mice on a
C57BL/6 background were kindly provided by Fred Alt
(Harvard Medical School and Center for Blood Research,
Boston, Massachusetts, USA). TLR4-deficient mice were
generated as described previously (24). Littermate wild-
type or heterozygous mutant mice were used for experi-
ments as control mice. All mice were housed in a specif-
ic pathogen-free facility at the Research Institute for
Microbial Diseases, Osaka University (Osaka, Japan).
Histopathological analysis. Paraffin-embedded colon
samples were sectioned and stained with H&E. Trans-
verse sections are shown in the figures. Severity of coli-
tis was evaluated by the standard scoring system as
described previously (25). Each region of the colon
(cecum; ascending, transverse, and descending colon;
and rectum) was graded semiquantitatively from 0 (no
change) to 5 (most severe change). The grading repre-
sents an increasing incidence and degree of inflamma-
tion, goblet cell loss, crypt abscesses and ulceration,
and fibrosis in the lamina propria. The summation of
the score for each segment of the colon provides a total
disease score per mouse (0–25) where: 0 indicates no
change; 1–5, mild disease; 6–10, mild-moderate; and
11–20, severe. No mice in these studies had a score
above 20 because such severe disease results in death.
The scoring was performed blinded manner by two
independent investigators. The colitis scores were eval-
uated using the program Statview II (SAS Inc., Cary,
North Carolina, USA). P values less than 0.05 were
assumed to be statistically significant.
Cytokine production by macrophages. Mice were intraperi-
toneally injected with 2 ml of 4% thioglycollate medium
(Sigma-Aldrich, St. Louis, Missouri, USA). Three days
later, peritoneal exudate cells were isolated from the
peritoneal cavity by washing with ice-cold HBSS
(Sigma-Aldrich). Cells were cultured for 2 h and washed
with HBSS to remove nonadherent cells. Adherent
monolayer cells were used as peritoneal macrophages.
Peritoneal macrophages were cultured with 10 ng/ml of
LPS (Escherichia coli serotype O55:B5), 1 µM of CpG
DNA, or 50 U/ml of IFN-γ(Genzyme, Cambridge, Mass-
achusetts, USA) plus LPS for 24 h. Concentrations of
TNF-α, IL-6, and IL-12p40 in the culture supernatants
were measured by ELISA according to the manufactur-
er’s instructions (Genzyme; Endogen Inc., Woburn,
Massachusetts, USA). Production of nitric oxide was
measured by the Greiss method using a NO2/NO3Assay
Kit (DOJINDO, Kumamto, Japan).
Cytokine production by dendritic cells isolated from the large
intestine. The large intestine was removed aseptically.
After removal of colonic patches, the large intestine was
washed with RPMI-1640 on ice and stirred with
RPMI-1640 containing 20 mM HEPES, 50 µg/ml of gen-
tamycin, 100 U/ml penicillin, 100 µg/ml streptomycin,
10% FCS, 5 mM EDTA for 60 min at 37°C. The tissues
were then digested with collagenase D (400 U/ml;
Boehringer Mannheim GmbH, Mannheim, Germany) in
RPMI-1640 containing 10% FCS for 30 min at 37°C and
further incubated in the presence of 5 mM EDTA for 5
min. The isolated cells were purified by centrifugation
through a 14.5% Accudenz gradient (Accurate Chemical
& Scientific Corp., Westbury, New York, USA). Cells were
further incubated with mouse CD11c microbeads (Mil-
tenyi Biotech, Bergisch Gladbach, Germany) and select-
ed on autoMACS separation columns (Miltenyi
Biotech). The purified dendritic cell fractions were more
than 95% CD11c+. These cells (105) were seeded on
96-well plate and cultured in the presence or absence of
The Journal of Clinical Investigation| May 2003| Volume 111|Number 9
triggering mucosal inflammation could be “intestinal
microflora,” commensal microorganisms resident on
the mucosal surface (7). Therefore, we propose the fol-
lowing hypothesis: TLRs expressed on myeloid cells rec-
ognize microbial components of the mucosal microflo-
ra and activate myeloid cells to produce inflammatory
cytokines including IL-10, IL-12, and TNF-α. In normal
conditions, IL-10, which is produced by myeloid cells
and regulatory T cells, suppresses the activity of myeloid
cells. In the absence of IL-10 signaling, however, myeloid
cells are abnormally activated through TLRs and pro-
duce increased levels of inflammatory cytokines. Among
these, IL-12 induces the development of colitis through
the generation of aberrant Th1 cells.
Some of TLR4/Stat3 double-mutant mice developed
colitis at old age, indicating that other TLRs may be
involved in the initiation of pathogenesis. TLR9, which
induces potent Th1 responses through recognition of
CpG DNA in vivo, is one such candidate (50). CpG
DNA induced increased production of inflammatory
cytokines, including IL-12p40, in TLR4/Stat3 double-
mutant macrophages (our unpublished data). There-
fore, we speculate that constitutive exposure of micro-
bial components such as CpG DNA may eventually
prime myeloid cells to produce increased level of
inflammatory cytokines in the TLR4/Stat3 double-
mutant mice. Even in the absence of LPS signaling, the
abnormal activity of myeloid cells may eventually lead
to the induction of pathological Th1 responses in the
double-mutant mice. It should be noted, however, that
the level of the CpG DNA/TLR9–mediated IL-12p40
production by the double-mutant macrophages was
lower when compared with the Stat3 mutant
macrophages, which may explain one of the causes for
the improvement of colitis in the double-mutant mice
(our unpublished data). In addition to TLR9, TLR5,
which has been shown to be expressed on the basolat-
eral side of intestinal epithelia and recognize bacterial
flagellin, may also be involved in the initiation of
inflammation cascade in the large intestine (51, 52).
One of the other interesting findings of the present
study is the demonstration of the accumulation of den-
dritic cells and macrophages in the disease site of Stat3
mutant mice (Figure 10). These myeloid lineages of
mucosal APCs produced increased level of inflammato-
ry cytokines such as IL-12p40 and IL-6. It should also
be noted that colocalization of mucosal dendritic cells
and CD3+T cells was directly demonstrated in the large
intestine of Stat3 mutant mice with colitis. Further-
more, intestinal dendritic cells have been shown to
extend their dendron to the lumen side of the intestine
(53).Thus, the direct communication between the den-
dritic cell/T cell clusters and outside environments,
including the commensal microflora, may occur
through TLRs and microbial components, respectively,
at the intestinal mucosa, which instructs the initiation
of large-intestinal inflammation in Stat3 mutant mice.
Finally, we demonstrated that IL-10–induced expres-
sion of SOCS3 is dependent on Stat3 in macrophages
(Figure 9, a–f). SOCS3 has been shown to inhibit the
LPS-induced expression of TNF-α, IL-6, and iNOS in
macrophages (32). Our present results show that the
severe reduction in the expression of SOCS3 well corre-
lates with the impaired IL-10–mediated suppression of
the LPS-induced IL-12p40 expression in Stat3 mutant
macrophages (Figure 9). Interestingly, expression of
IL-12p40 mRNA induced by 2-h LPS stimulation was
comparable between control and Stat3 mutant macro-
phages (Figure 9b). IL-12p40 protein production during
24-h stimulation, however, was significantly increased in
Stat3 mutant mice (Figures 2, 3, 5, 7). These findings
may indicate that the initial LPS response was equivalent
between control and Stat3 mutant macrophages, but
after several hours of LPS stimulation, the difference
may arise for the induction of IL-10–inducible genes
associated with suppressor activity. Our findings suggest
that one candidate would be IL-10–mediated expression
of SOCS3 that suppresses macrophage activity. One
must emphasize, however, that several other genes are
also induced in response to IL-10 in macrophages (ref.
31 and our unpublished data). Therefore, there is a pos-
sibility that an additional gene product other than
SOCS3 may also be responsible for the IL-10–mediated
suppression of macrophages. Precise analysis of the
mechanism by which IL-10 suppresses activity of
myeloid cells, especially in response to LPS stimulation,
would be an interesting issue, which needs to be eluci-
dated in the future.
We thank Irmgard Förster, Kenji Sekikawa, Jeanne
Magram, and Fred Alt for providing LysMcre mice, TNF-
α-, IL-12-, and RAG2-deficient mice, respectively. We also
thank T. Tsujimura for histopathological analysis, N.
Okita for technical assistance, and E. Horita for secre-
tarial assistance. This work was supported by grants
from Special Coordination Funds, the Ministry of Edu-
cation, Culture, Sports, Science and Technology, and the
Japan Research Foundation for Clinical Pharmacology.
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