Inflammation-induced tumorigenesis in the colon is
regulated by caspase-1 and NLRC4
Bo Hua,b,1, Eran Elinava,1, Samuel Hubera, Carmen J. Boothc, Till Strowiga, Chengcheng Jina,d,
Stephanie C. Eisenbartha,e, and Richard A. Flavella,f,2
aDepartment of Immunobiology,bDepartment of Molecular Biophysics and Biochemistry,cSection of Comparative Medicine,dDepartment of Cell Biology,
eDepartment of Laboratory Medicine, andfHoward Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520
Contributed by Richard A. Flavell, November 9, 2010 (sent for review September 10, 2010)
Chronic inflammation is a known risk factor for tumorigenesis, yet
the precise mechanism of this association is currently unknown. The
(NLR) family members, has recently been shown to orchestrate
multiple innate and adaptive immune responses, yet its potential
azoxymethane and dextran sodium sulfate colitis-associated co-
lorectal cancer model, we show that caspase-1–deficient (Casp1−/−)
mice have enhanced tumor formation. Surprisingly, the role of cas-
pase-1 in tumorigenesis was not through regulation of colonic in-
flammation, but rather through regulation of colonic epithelial cell
proliferation and apoptosis. Consequently, caspase-1–deficient mice
demonstrate increased colonic epithelial cell proliferation in early
stages of injury-induced tumor formation and reduced apoptosis in
advanced tumors. We suggest a model in which the NLRC4 inflam-
masome is central to colonic inflammation-induced tumorformation
through regulation of epithelial cell response to injury.
colon cancer|inflammation-induced colorectal cancer|NLR family, pyrin
domain containing 3
is poorly understood (1). Chronic inflammation is a known risk
factor for tumorigenesis, and epidemiological data suggest that
up to 15% of human cancer incidence is associated with inflam-
mation (2, 3). Inflammation-induced colorectal cancer develops
in patients with chronic inflammatory bowel disease, with risk
estimated to increase by 0.5 to 1% per year after 8 to 10 y of in-
flammatory bowel disease (4). The nature of this strong associa-
tion is largely unknown, and suggested mechanisms include
chronic formation of reactive oxygen species (5) and tumorigen-
esis induced by chronic epithelial exposure or inflammatory
stimuli, such as IL-6 and TNF-α (6–9). The importance of in-
flammation is further highlighted by the dependence of tumor
growth and progression on the activation of NF-κB by the classic
inhibitor of nuclear factor kappa-B kinase-dependent pathway,
which is crucial for tumor growth and progression (10).
Inflammasomes are multiprotein complexes formed by several
members of the NOD-like receptor (NLR) family, procaspase-1,
and the adaptor protein ASC, originally described by Tschopp and
colleagues to be central mediators of the innate immune response
(11). Certain NLR family members can mediate inflammasome
assemblyinresponse todiverse danger signals,includingpathogen-
acid crystals, and aluminum hydroxide (11, 12). Upon activation,
caspase-1 cleaves the proinflammatory cytokines IL-1β and IL-18,
resulting in secretion of their mature forms (11). The NLRP3 (also
known as CIAS1 and NALP3) inflammasome is the most thor-
oughly characterized inflammasome; it can be activated by a num-
ber of chemically and structurally diverse triggers (12–14) and has
recently been shown to orchestrate multiple innate and adaptive
immune responses (12, 15).
NLRC4 belongs to the NLR family with a characteristic N-
terminal CARD domain, a central NACHT domain, and C-
olorectal cancer is one of the most common forms of fatal
cancer in the world, yet its underlying molecular pathogenesis
terminal leucine-rich repeats. The NLRC4 inflammasome has
been demonstrated to be important in host defense against
a number of Gram-negative bacterial pathogens, such as Pseu-
domonas, Salmonella, and Shigella (16–18). Specifically, the
NLRC4 inflammasome is activated by flagellin and components
of the Type III secretion system of pathogenic Gram-negative
bacteria (18–20). Unlike other inflammasomes, NLRC4 can ac-
tivate caspase-1 in an ASC-dependent or -independent manner
(18, 21). Like other described inflammasomes, NLRC4 inflam-
masome formation results in secretion of IL-1β and IL-18, but can
also induce caspase-1–mediated cell death (18, 22). Furthermore,
NLRC4 has also been suggested to participate in apoptosis
pathways downstream of p53, as NLRC4 gene expression induced
by p53 contributes to p53-dependent apoptosis in several human
cell types (23, 24).
In addition to orchestrating multiple innate and adaptive im-
mune responses upon encountering pathogens, the NLRP3
inflammasome has also been demonstrated to take part in IL-1β–
dependent adaptive immune responses against dying tumor cells
(25). ATP released from chemotherapy-treated tumors activates
the NLRP3 inflammasome in dendritic cells, resulting in an ef-
fective CD8+T-cell response directed against the tumor (25).
However, the potential role of inflammasome signaling in the
initiation and progression of inflammation-induced cancer has not
been previously studied. Furthermore, mice deficient in MyD88,
a central component in the signaling pathways downstream of the
majorityofToll-likereceptors (TLR),exhibitreducedtumor loads
in a model of colon cancer (26). An explanation for this observa-
tion is that reduced colonic inflammation because of lack of TLR
signaling following bacterial exposure decreases tumor formation
in the colon. However, because MyD88 is also an essential com-
ponent of the signaling cascade downstream of the IL-1 receptor
family, the phenotype could be solely or partly explained by re-
duction of IL-1β and IL-18 signaling in the absence of a contribu-
tion of TLRs. We decided, therefore, to test this hypothesis in
caspase-1–deficient mice that lack the ability to produce IL-1β and
IL-18 and that should therefore have decreased inflammation-
induced carcinogenesis. Support for the involvement of IL-18
came from a recent article in which IL-18−/−mice were shown to
develop severe colitis and also increased tumorigenesis compared
with WT, suggesting that enhanced tumorigenesis in TLR dys-
functional mice is linked to signaling through the IL-18 receptor
(27). The role of inflammasome components in the pathogenesis
of colonic autoinflammation remains controversial. Three recent
reports suggested that deficiency in caspase-1, ASC, or NLRP3 in
Author contributions: B.H., E.E., S.C.E., and R.A.F. designed research; B.H., E.E., S.H., T.S.,
and C.J. performed research; C.J.B. contributed new reagents/analytic tools; B.H., E.E.,
S.H., C.J.B., T.S., C.J., and S.C.E. analyzed data; and B.H., E.E., S.C.E., and R.A.F. wrote
The authors declare no conflict of interest.
1B.H. and E.E. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| December 14, 2010
| vol. 107
| no. 50
mice was associated with an increased severity of chemically in-
duced colitis and related tumorigenesis, suggested to be mediated
in part by a defect in repair of the intestinal mucosa (28–30).
However, opposite results were found in another study using the
same chemically induced colitis model, demonstrating an amelio-
rated severity of colitis in NLRP3-deficient mice as a result of
decreased levels of proinflammatory IL-1β (31). Differences be-
tween these studies may stem from methodological differences in
the induction protocols as well as by inherent differences between
variables, such as the composition of the intestinal flora.
In this article, we demonstrate a unique and surprising link
between the inflammasome and colorectal inflammation-induced
tumorigenesis. In contradiction to our hypothesis, we show that
caspase-1–deficient mice have enhanced tumor formation in the
azoxymethane (AOM) and dextran sodium sulfate (DSS) colitis-
role of caspase-1 in tumorigenesis is mediated through direct and
profound effects on colonic epithelial cell proliferation and apopto-
sis, rather than through regulation of colonic inflammation. Fur-
thermore, we demonstrate that increased tumorigenesis is mediated
through the NLRC4 inflammasome, rather than through NLRP3.
Results and Discussion
colorectal cancer, we used the AOM-DSS model, in which sys-
temically administered AOM induces colon tumorigenesis in mice
induced with chronic DSS colitis. In the AOM-DSS inflammation-
a significant increase in tumor numbers and tumor load compared
heavy tumor load in 20% of Casp1−/−mice resulted in large bowel
obstruction and prevented the passage of a murine endoscope
(Fig. 1C). Histopathologically, tumors from Casp1−/−mice were
indistinguishable from those of WT mice, where all tumors were
adenocarcinoma (Fig. 1D, I). However, tumors of Casp1−/−mice
appeared more aggressive with invasion (arrows) of tumor cells
below the muscularis mucosae (*) noted in 75% of Casp1−/−mice,
compared with 14% invasion in WT group in advanced stages of
disease (day 200) (Fig. 1D, II). No evidence of distal metastasis in
the lungs, liver, spleen, or bone marrow was noted in mice that
were killed on day 120 of the CAC regimen.
One potential mechanism contributing to the increased tu-
morigenesis in Casp1−/−mice is increased colonic inflammation in
these mice. To test this possibility, we used the acute DSS colitis
model that employs a single 7-d-long cycle of DSS exposure (10).
We did not observe any differences in mass loss (Fig. 2A), colo-
noscopic inflammation severity score (Fig. 2B), and histopatho-
logical morphology (Fig. 2 C and D), between Casp1−/−and WT
when substantial inflammation was notable both endoscopically
and pathologically. Likewise, when chronic DSS colitis was in-
duced by three 5-d cycles of DSS after AOM injection, no sig-
nificant difference in chronic colonic inflammation were noted
between Casp1−/−and WT mice (Fig. 2E). In addition, no sig-
nificant changes were noted in both mRNA and protein levels of
inflammatory cytokine in both acute and chronic DSS colitis (Fig.
S1). This surprising result prompted us to investigate whether the
dramatic enhancement in tumor formation observed in Casp1−/−
mice may be caused by intrinsic effects in the colonic epithelial
cells, rather than by indirect effects stemming from the degree
of surrounding inflammation, as both hematopoietic and non-
hematopoietic cells in the colon express caspase-1 (32). To in-
vestigate which of these cellular compartments is mainly
responsible for the increased tumorigenesis in Casp1−/−mice, we
created bone marrow-chimeric mice, which were induced with
AOM-DSS inflammation-induced colon cancer. We found that
both WT bone marrow transplanted Casp1−/−mice and Casp1−/−
bone marrow transplanted WT mice did not recapitulate the
and tumor numbers/mouse in WT and caspase-1 KO (Casp1−/−) mice (n = 8). P values < 0.05 were considered statistically significant. The experiments were
repeated five times. (C) Representative colonoscopic appearance of WT and Casp1−/−mice colon on day 65 of AOM-DSS–induced color cancer. (D) Represen-
and less frequent in WT mice than Casp1−/−mice, where tumor foci were surrounded by abundant amounts of pale blue mucin. H&E staining; *muscularis
mucosae; m, mascularis externa. (Scale bars, 200 μm.)
Caspase-1 deficiency exacerbates AOM-DSS–induced colon cancer. (A) Schematic overview of the inflammation-induced cancer model. (B) Tumor load
| www.pnas.org/cgi/doi/10.1073/pnas.1016814108 Hu et al.
Casp1−/−phenotype (Fig. S2). These data suggest that both the
“radio-sensitive” hematopoietic compartment and the “radio-re-
sistant” epithelial compartment are required for the increased
tumorigenesis observed in Casp1−/−mice. However, involvement
of prolonged antibiotic treatment in this model, which in our
hands severely alters the microbiotic flora, results in increased
variability between experiments and limits the interpretation of
the bone-marrow transplantation results.
Enhanced Colon Epithelial and Tumor Cell Proliferation in Casp1−/−
Mice During Inflammation-Induced Carcinogenesis. One of the sug-
gested mechanisms to explain the initiation of colonic injury-
induced cancer is repetitive epithelial cell destruction and sub-
sequent regeneration, resulting in enhanced mutation (33, 34). To
investigate this possibility, we analyzed the proliferation and sur-
vival of intestinal epithelial cells at day 15 of the CAC regimen in
Casp1−/−and WT mice. At this stage, when inflammation is at its
mice (Fig. 3A); however, small foci of crypt hyperplasia/dysplasia
(Fig. 3D) were observed in both strains of mice. Immunohisto-
chemistrywithKi67 (Fig.3B) andBrdU(Fig.3C),bothmarkersof
cellular proliferation, revealed significant increases in colon crypt
cell proliferation in Casp1−/−mice compared with WT mice.
no increase in the number of Ki67+cells (Fig. 3E) and a moderate
increase in the number of BrdU+cells (Fig. 3F) in Casp1−/−mice
when compared with WTmice. Inadvancedtumorsat day 65 (Fig.
3G), the numbers of both Ki67+cells (Fig. 3H) and BrdU+cells
(Fig. 3I) were higher in Casp1−/−mice compared with WT mice.
Quantification of the immunohistochemical data validated the
statistically significant increase in BrdU+colonic epithelial cells in
Casp1−/−mice at baseline, as well as an increase in Ki67+and
BrdU+cells inbothcolonicand tumortissuein advanced stagesof
the CAC regimen (Fig. S3). This validation suggests that a signifi-
cantly increased proportion of cells in Casp1−/−tumors were ac-
tively proliferating compared with WT tumors. This difference
may stem from caspase-1–dependent cleavage of a yet un-
recognized negative regulator of cell proliferation. To further ex-
amine the possibility that epithelial cell survival and apoptosis also
differ between Casp1−/−and WT mice, we performed in situ
TUNEL staining on colon sections of mice in the early and late
stages of AOM-DSS experiments. We noted no significant differ-
ences in TUNEL-positive cells in the colon between Casp1−/−and
advanced Casp1−/−tumors (day 65) exhibited a significantly re-
duced percentage of TUNEL-positive cells compared with WT
mice (Fig. 3J, Right).
To address the question whether differences in antitumor im-
mune responses contribute to the increased tumor formation in
Casp1−/−mice, we stained colon sections frommice withadvanced
AOM-DSS–induced colon tumors (day 65 and day 200) with anti-
bodies against CD3, Myeloperoxidase (MOP) and NK1.1. No dif-
ferences were noted between the WT and KO mice in regard
3.193 CD3+cells/field;MOP-1 staining: WT tumors: 18.50 ± 1.190
MOP-1+cells/field, Casp1−/−tumors 19.42 ± 1.694 MOP-1+cells/
field; mice, n ≥ 6, day 200 of the CAC regimen) and very rare
NK1.1+cells were notedin all samples, which is in agreement with
Increased Tumorigenesis in Casp1−/−Mice Is Mediated by the NLRC4
Inflammasome. Increased tumor formation in Casp1−/−mice sug-
gests that inflammasome activation participates in pathways con-
trolling the development and growth of tumors. The two most
widely studied inflammasomes are the NLRP3 and NLRC4
inflammasomes. Both have been shown to participate in innate
immune response to exogenous and endogenous “danger signals,”
such as ATP, crystals, and Gram-positive and -negative bacteria.
In addition, the NLRC4 inflammasome has been suggested to
participate in antiapoptosis pathways downstream of p53 (23,
24). To investigate the possible involvement of these NLRs in
inflammation-induced cancer formation, we first examined the
expression of caspase-1, NLRP3, and NLRC4 mRNAs in the ep-
4A). Caspase-1 and NLRC4 were found to be relatively highly
expressed in both colonic epithelial cells and CD45+hematopoi-
etic cells in the colon as compared with hypoxanthine phosphor-
ibosyltransferase (HPRT). In contrast, NLRP3 expression was
primarily restricted to the hematopoietic compartment. To eval-
uate their contribution to the increased tumor formation in
tumor formation were observed between NLRP3−/−and WT mice
(Fig. 4B). In contrast, NLRC4−/−mice had significantly increased
Similarly to Casp1−/−mice, tumors of NLRC4−/−mice appeared
more aggressive with invasion (arrows) of tumor cells below the
muscularis mucosae (*) noted in 66% of NLRC4−/−mice, com-
pared with no invasion observed in WT group in advanced stages
of disease (day 80) (Fig. S4). These results suggest that the dif-
ferences in tumor formation noted in Casp1−/−mice are mediated
through the NLRC4 inflammasome rather than the NLRP3
inflammasome. When DSS colitis wasinduced in NLRC4−/−mice,
again no differences in inflammation severity were noted (Fig. 4 D
and E, measuring mass loss or colonoscopy inflammation severity,
respectively), further supporting the hypothesis that intrinsic,
during acute DSS colitis. There were no significant difference in percent-mass
change (A), colonoscopy inflammation severity score (B), severity of histo-
pathological morphology (C) (H&E staining), or histopathology parameters
for edema, inflammation, ulceration or overall severity of injury (D) between
WT and Casp1−/−mice. (Scale bars in C, 200 μm.) (E) Likewise, the severity of
chronic DSS colitis was similar between WT and Casp1−/−mice. All experi-
ments were repeated twice.
No differences in inflammation between WT and Casp1−/−mice
Hu et al.PNAS
| December 14, 2010
| vol. 107
| no. 50
rather than inflammation-related changes, account for the differ-
ences in tumor formation in inflammasome-deficient mice. As
NLRC4 is implicated in p53-dependent apoptosis, it may provide
and the changes in proliferation and apoptosis noted in epithelial
activated by Gram-negative bacteria may point to its involvement
in modulation of the colonic tumor formation by events involving
the gut commensal bacterial flora (35). Future studies should aim
and caspase-1 that may lead to enhanced tumorigenesis in both.
Our results differ from the report by Allen et al. which suggested
(28). Difference between the studies may stem from methodo-
logical differences in the induction protocolsas well as by inherent
differences between variables, such as the composition of the
To further study the molecular mechanism of Casp1−/−epi-
thelial tumor transformation, we studied caspase-1 mRNA ex-
pression levels in normal colon tissue and colon tumors from WT
mice. We observed a significant reduction in caspase-1 mRNA
expression level in the tumor compared with colon tissue, sug-
gesting thatlackofcaspase-1 mayplayarole intumorprogression
(Fig. 4F). Furthermore, isolated colonic epithelial cells from ei-
ther distal or proximal colons of mice with AOM-DSS–induced
tumors featured a significant reduction in caspase-1 mRNA ex-
pression levels compared with naive mice, suggesting that re-
duction in caspase-1 expression specifically in epithelial cells may
play a role in tumorigenesis (Fig. 4G). Similar results in humans
showed that caspase-1 is down-regulated in human colon ade-
nocarcinomas both at the mRNA and protein level (32). Similarly
to Casp1−/−mice, NLRC4−/−mice featured significantly en-
hanced proliferation in both steady state and the early phase of
inflammation-induced tumor formation and reduced apoptosis in
tumors (Fig. S5).
and BrdU immunohistochemistry at day 15 (A–F) and at day 65 (G–I), and TUNEL-positive cells in colons (day 8) and tumors (day 65) in WT and Casp1−/−mice
given AOM-DSS. The majority of crypts at day 15 appeared normal (A); however, there were increased Ki67+(B) and BrdU+(C) crypt epithelial cells in Casp1−/−
mice compared with WT mice. In foci of crypt hyperplasia/dysplasia (D), there were no significant difference in the number of Ki6+cells (E) and a moderate
increase in the number of BrdU+cells (F) in Casp1−/−compared with WT mice. At day 65 there were numerous large colon adenocarcinomas (G) with increased
numbers of Ki67+tumor cells (H) and BrdU+tumor cells (I) in Casp1−/−compared with WT mice. (A, D, G: H&E staining; B, C, E, F, H, and I: DAB, Hematoxalyn)
(Scale bars, 200 μm). (J) The number of TUNEL-positive cells in colon is similar in WT compared with Casp1−/−mice. In contrast, there is reduced cell death of
tumor tissue in Casp1−/−mice (average cells counted: WT: 34.27 ± 8.964 positive cells/293.6 ± 86.39 total cells per field; Casp1−/−: 16.87 ± 4.533 positive cells/
364.2 ± 98.70 total cells per field). DNA fragmentation in WT and Casp-1−/−mice was determined on either whole-colon sections on day 8 of AOM-DSS model
or tumor tissues at day 65. Error bars represent ± SEM, P < 0.001. The experiments were repeated two to three times.
Enhanced colon epithelial and tumor cell proliferation in Casp1−/−mice during inflammation-induced colorectal cancer. Representative H&E, Ki67,
| www.pnas.org/cgi/doi/10.1073/pnas.1016814108Hu et al.
In conclusion, our results support a model in which caspase-1 is
central in the prevention of colonic inflammation-induced tumor
formation through regulation of the epithelial cell response to in-
jury. These effects are mediated through the NLRC4 inflamma-
some. Lack of such activation results in intrinsic changes in
epithelial gene-expression programming, including enhanced pro-
liferation, reduced apoptosis in tumor tissue, and overall enhanced
tendency for tumor formation. In agreement with our findings,
inflammasome-deficient mice were recently suggested to feature
contrast to the recent published reports (28, 29). Thus, our results
tumorigenesis in the absence of caspase-1 or NLRC4 activity.
Further deciphering of these mechanisms may enhance our un-
derstanding of events leading to inflammation-initiated tumor for-
mation and may enable the recognition of new therapeutic targets.
Materials and Methods
Experimental Animals. All mice were cared for in accordance with institutional
animal care and use committee-approved protocols at the Yale University
School of Medicine animal facility.
Tumor Induction and Analysis. Age- and sex-matched cohoused 6- to 8-wk-old
mice (WT, Casp1−/−, NLRC4−/−, and NLRP3−/−mice on C57BL/6 background)
were injected with AOM (Sigma) intraperitoneally at a dose of 12.5 mg/kg
body weight. After 5 d, mice were treated with 2.5% DSS (MP Biomedicals)
(M.W. 36,000–50,000 Da) in the drinking water for 5 d, then followed by
16 d of regular water. This cycle was repeated twice (10).
DSS Colitis. To induce acute colitis, 6- to 8-wk-old mice (WT, Casp1−/−, and
NLRC4−/−mice on C57BL/6 background) were treated with 2% DSS (MP
Biomedicals) (M.W., 36,000–50,000 Da) in the drinking water for 7 d fol-
lowed by regular access to water. In the chronic colitis experiments, DSS was
administered in three 5-d cycles, as is depicted in Fig. 1A.
Endoscopic Procedures. Colonoscopy was performed at indicated time points
to monitor for severity of colitis and tumorigenesis according to murine
endoscopic index of colitis severity system (36). A detailed endoscopic pro-
cedure is described in SI Materials and Methods.
Histopathology. After processing, colons were embedded lengthwise in
the level of the lumen and then the next 5-μm section was stained with H&E,
followed by placement of coverslips by routine methods. Colons were
evaluated and were assigned scores by investigators blinded to experimental
manipulation (37). A detailed histopathology procedure is described in SI
Materials and Methods.
epithelial cells and CD45+cells. The levels of the indicated mRNAs were quantitated by real-time PCR and normalized to the level of HPRT mRNA. (B)
Inflammation–induced tumor formation is similar in WT and NLRP3−/−mice (n ≥ 10). The experiments were repeated three times. (C) NLRC4−/−mice feature
enhanced inflammation-induced colon cancer compared with WT mice (n ≥ 6); P < 0.05 was considered statistically significant. The experiments were re-
peated twice. Severity of DSS colitis in WT and NLRC4−/−mice, as evident by (D) mass change and (E) inflammation colonoscopy severity score. The
experiments were repeated twice. (F) Caspase-1 mRNA expression in normal and colon and adjacent tumors from WT mice. mRNA levels were assessed by
real-time PCR and normalized to the level of HPRT (n ≥ 5). The experiment was repeated twice. P < 0.05 was considered statistically significant. (G) Caspase-1
mRNA expression in naive and AOM-DSS treated proximal and distal colonic epithelial cells from WT mice. mRNA levels were assessed by real-time PCR and
normalized to the level of HPRT (n ≥ 8). The experiment was repeated twice. P < 0.05 was considered statistically significant.
Caspase-1–mediated tumor enhancement is mediated by the NLRC4 inflammasome. (A) Caspase-1, NLRP3, and NLRC4 mRNA expression in colon
Hu et al. PNAS
| December 14, 2010
| vol. 107
| no. 50
Isolation of Epithelial Cells and Hematopoietic Cells from the Intestine. Epi- Download full-text
thelial cells and hematopoietic cells were isolated from the freshly dissected
colon by HBSS/EDTA shake and centrifugation. Detailed isolation procedures
are described in SI Materials and Methods.
Immunohistochemistry. Paraffin-embedded sections were dehydrated and
incubated with the following antibodies: anti-BrdU (1:4,000; Sigma), anti-
Ki67 (1:100, Thermo; Lab Vision), anti-CD3 (1:400; Biocare Medical), anti-
myeloperoxidase (1:100, Thermo; Lab Vision), anti-Nk1.1 (1:100, Novus Bio-
logicals). DAKO EnVision System was used for detection. All sections were
counterstained with hematoxylin. BrdU, TUNEL assay, and quantifications are
detailed in SI Materials and Methods.
Colon Culture and ELISA. Sections of 1 cm of the proximal colon were cut, re-
moved of feces, washed with PBS, and then cut half longitudinally. The colon
sections were placed into culture in RPMI-1640 media (Sigma) with high con-
centration of penicillin and streptomycin and cultured at 37 °C with 5% CO2.
Supernatants were harvested after 24 or 36 h, and the concentration of cyto-
kine was determined by ELISA. Antibody pairs were purchased from BD Phar-
mingen (IFN-γ) and SouthernBiotech (IL-17), and ELISA was performed
according to the manufacturer’s protocols.
RNA Analysis. Total RNA was extracted from colon tissue, tumors, and purified
cellsusingTRIzol Reagent. SuperScriptIIReverseTranscriptase (Invitrogen)was
PCR Mater Mix and TaqMan Gene Expression Assays (Applied Biosystems) was
performed on a 7500 Fast Real-time PCR system machine(AppliedBiosystems).
Statistical Analysis. Data are expressed as mean ± SEM. Differences were an-
alyzed by Student’s t test and one way ANOVA and post hoc analysis for
multiple group comparison. P values < 0.05 were considered significant.
ACKNOWLEDGMENTS. We thank Lauren Zenewicz, Adam Williams, Tony
Ferrandino, Jon Alderman, Elizabeth Eynon, Anthony Rongvaux, William
O’Connor, Chozhavendan Rathinam, Yasushi Kobayashi, Jorge Henao Mejia,
Paula Licona Limon, and all members of the R.A.F. laboratory; Namiko Hoshi
for technical help and scientific discussion; Fran Manzo for manuscript prep-
aration; Ruslan Medzhitov, David Schatz, and Patrick Sung for scientific dis-
cussion; and the Yale Research Histology Laboratory in the Department of
Pathology and the Yale Mouse Research Histopathology in the Section of
Comparative Medicine for histology and immunohistochemistry help. This
study was funded in part by a Cancer Research Institute postdoctoral fellow-
ship and by a supplemental grant by the United States-Israel Educational
Foundation (to E.E.), Deutsche Forschungsgemeinschaft Hu1714/1-1 and the
James Hudson Brown-Alexander Brown Postdoctoral Fellowships (to S.H.),
National Institutes of Health Grant T32Hl007974 (to S.C.E.), and the Howard
Hughes Medical Institute (R.A.F.).
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