IL-18 Production Downstream of the Nlrp3 Inflammasome Confers Protection against Colorectal Tumor Formation

Article (PDF Available)inThe Journal of Immunology 185(8):4912-20 · October 2010with38 Reads
DOI: 10.4049/jimmunol.1002046 · Source: PubMed
Abstract
Colorectal cancer is a leading cause of cancer-related deaths worldwide. Chronic inflammation is recognized as a predisposing factor for the development of colon cancer, but the molecular mechanisms linking inflammation and tumorigenesis have remained elusive. Recent studies revealed a crucial role for the NOD-like receptor protein Nlrp3 in regulating inflammation through the assembly of proinflammatory protein complexes termed inflammasomes. However, its role in colorectal tumor formation remains unclear. In this study, we showed that mice deficient for Nlrp3 or the inflammasome effector caspase-1 were highly susceptible to azoxymethane/dextran sodium sulfate-induced inflammation and suffered from dramatically increased tumor burdens in the colon. This was a consequence of markedly reduced IL-18 levels in mice lacking components of the Nlrp3 inflammasome, which led to impaired production and activation of the tumor suppressors IFN-γ and STAT1, respectively. Thus, IL-18 production downstream of the Nlrp3 inflammasome is critically involved in protection against colorectal tumorigenesis.

Figures

The Journal of Immunology
IL-18 Production Downstream of the Nlrp3 Inflammasome
Confers Protection against Colorectal Tumor Formation
Mohammad Hasan Zaki,* Peter Vogel,
Mathilde Body-Malapel,
Mohamed Lamkanfi,
x,{
and
Thirumala-Devi Kanneganti*
Colorectal cancer is a leading cause of cancer-related deaths worldwide. Chronic inflammation is recognized as a predisposing
factor for the development of colon cancer, but the molecular mechanisms linking inflammation and tumorigenesis have remained
elusive. Recent studies revealed a crucial role for the NOD-like receptor protein Nlrp3 in regulating inflammation through the
assembly of proinflammatory protein complexes termed inflammasomes. However, its role in colorectal tumor formation remains
unclear. In this study, we showed that mice deficient for Nlrp3 or the inflammasome effector caspase-1 were highly susceptible to
azoxymethane/dextran sodium sulfate-induced inflammation and suffered from dramatically increased tumor burdens in the colon.
This was a consequence of markedly reduced IL-18 levels in mice lacking components of the Nlrp3 inflammasome, which led to
impaired production and activation of the tumor suppressors IFN-g and STAT1, respectively. Thus, IL-18 production downstream
of the Nlrp3 inflammasome is critically involved in protection against colorectal tumorigenesis. The Journal of Immunology,
2010, 185: 4912–4920.
C
olorectal cancer is one of the leading causes of cancer-
related deaths. Patients with inflammatory bowel dis-
eases (IBDs), most commonly Crohn’s disease and ul-
cerative colitis, are at increased risk of developing colorectal
cancer (1–3). Indeed, IBD is considered the third most important
risk factor for the development of colorectal cancer (4). Although
the precise molecular mechanism of IBD-related colorectal tumor
formation is not clearly understood, existing studies suggest that
chronic inflammation primes the mucosal tissue in the gut for
increased cell proliferation, angiogenesis, and tumor invasiveness
(5). In this regard, proinflammatory cytokines such as IL-1b, IL-6,
IL-18, TNF-a, and IFNs have been demonstrated to exert key
roles in inducing gut inflammation and colorectal tumor formation
(6–8). The synthesis and secretion of these cytokines is controlled
by transcription factors of the STAT, NF-kB, and AP-1 families
(6). Notably, recent evidence suggests that inhibition of NF-kB
reduces tumorigenesis (9, 10).
NF-kB activation and induction of additional inflammatory
signaling pathways is initiated by engagement of pathogen rec-
ognition receptors of the TLR and NOD-like receptor (NLR)
families (11, 12). TLRs are membrane-bound receptors that detect
pathogen-associated molecular patterns in the extracellular milieu
(13). The role of TLRs in the recruitment of immune cells at
mucosal surfaces and in protection against tumorigenesis in the
gut is well established. For example, TLR5 activation in a mouse
xenograft model of human colon cancer elicited powerful antitu-
mor activity (14). In addition to TLRs, several members of the
cytosolic NLR family have been identified as key regulators of
cytokine production (11). The NLR proteins NOD1 and NOD2
mediate activation of NF-kB and MAPKs in response to the cy-
tosolic presence of peptidoglycan fragments. In contrast, the NLR
protein Nlrp3 (also referred to as Nalp3/CIAS1/cryopyrin) is in-
volved in activation of the cysteine protease caspase-1 (15).
Homotypic interactions between the pyrin domain in the N termi-
nus of Nlrp3 and the bipartite adaptor protein apoptosis-associated
speck-like protein containing CARD (ASC) bridge the association
of caspase-1 to Nlrp3 in a large protein complex known as the
“inflammasome” (16). Activated caspase-1 processes the cytosolic
precursors of the related cytokines IL-1b and IL-18, thus allowing
secretion of the biologically active cytokines. Hence, mice lacking
caspase-1 are defective in the maturation and secretion of IL-1b
and IL-18 (17, 18). IL-1b participates in the generation of systemic
and local responses to infection, injury, and immunological chal-
lenges by generating fever , activ ating lymphocytes, and by pro-
moting leukocyte infiltration at sites of injury or infection. Binding
of IL-18 to the IL-18R complex triggers many of the signaling
pathways that are engaged by the IL-1R, including activation of
NF-kB, STAT1, and MAPKs (19, 20). IL-18 (previously known
as IFN-g–inducing factor) also promotes the production of IFN-g in
activated T cells and NK cells, thereby contributing to Th1 cell
polarization (8, 21, 22). Finally, IL-18 was shown to induce Fas
ligand production and the generation of multiple secondary proin-
flammatory cytokines, chemokines, cell adhesion molecules, and
NO species (23, 24).
The profound role of NLR-mediated inflammatory responses
in shaping the microenvironment during colitis-associated co-
lorectal tumorigenesis is starting to emerge. For instance, the NLR
family member NOD1 is important for protection against colitis-
associated colorectal tumor formation (25). Additionally, defective
activation of the Nlrp3 inflammasome was linked to increased
susceptibility to Crohn’s disease in patients (26). Moreover, the
*Department of Immunology and
Veterinary Pathology Core, St. Jude Children’s
Research Hospital, Memphis, TN 38105;
Department of Physiopathology of Inflam-
matory Bowel Diseases, INSERM Unite
´
995, Lille, France;
x
Department of Bio-
chemistry, Ghent University; and
{
Department of Medical Protein Research, Flan-
ders Institute of Biotechnology, Ghent, Belgium
Received for publication June 18, 2010. Accepted for publication August 11, 2010.
This work was supported in part by National Institutes of Health Grants AR056296
and AI088177, Cancer Center Support Grant CCSG 2 P30 CA 21765, and the
American Lebanese Syrian Associated Charities (to T.-D.K.). M.L. is supported by
the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.
Address correspondence and reprint requests to Dr. Thirumala-Devi Kanneganti,
Department of Immunology, St. Jude Children’s Research Hospital, MS 351, 570
St. Jude Place, Suite E7004, Memphis, TN 38105. E-mail address: thirumala-devi.
kanneganti@stjude.org
Abbreviations used in this paper: AOM, azoxymethane; ASC, apoptosis-associated
speck-like protein containing CARD; COX, cyclooxygenase; DSS, dextran sodium
sulfate; IBD, inflammatory bowel disease; NLR, NOD-like receptor; WT, wild-type.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002046
Nlrp3 inflammasome was recently shown to confer protection
against experimental colitis in mice (27–29). In this regard, mice
lacking the inflammasome components Nlrp3, ASC, or caspase-1
all presented with more severe clinical manifestations of colitis
and suffered from increased epithelial injury, bacterial invasion,
and death rates (27–29). The increased susceptibility to colitis was
correlated with defective IL-18 production in inflammasome-
deficient mice (27, 29). Despite the role of the Nlrp3 inflamma-
some in controlling colitis-associated inflammation, its roles in
controlling colitis-associated tumorigenesis and the relevant inflam-
masome effector pathways in this process have remained unclear.
To resolve these issues, we determined the rate of colorectal tumor
formation in Nlrp3
2/2
and Casp1
2/2
mice in the commonly used
azoxymethane (AOM)/dextran sodium sulfate (DSS) model. IL-18
production downstream of the Nlrp3 inflammasome was found to
exert a protective role against colorectal tumor formation. IL-
18–mediated activation and induction of the respective tumor
suppressors STAT1 and IFN-g may represent a potentially crit-
ical mechanism for Nlrp3-mediated resistance against colitis-
associated tumorigenesis.
Materials and Methods
Mice
Nlrp3
2/2
, ASC
2/2
,andCasp1
2/2
mice backcrossed to a C57BL/6 back-
ground for at least 10 generations have been described before (30). IL-18
2/2
mice were donated by Dr. Paul G. Thomas (St. Jude Children’s Research
Hospital). All mice were 8- to 10-wk-old males and were maintained in
a pathogen-free facility, and the animal studies were conducted under
protocols approved by the St. Jude Children’s Research Hospital Com-
mittee on the Use and Care of Animals.
Induction of colorectal cancer
Mice were injected i.p. with 10 mg/kg AOM (Sigma-Aldrich, St. Louis,
MO). After 5 d, 3% DSS (molecular mass, 36–40 kDa; MP Biologicals,
Solon, OH) was given in drinking water over 5 d followed by regular
drinking water for 2 wk. This cycle was repeated twice and mice were
sacrificed 4 wk after the last DSS cycle.
Histopathological analysis
Formalin-preserved sections of cecum and colon (proximal, middle, and
distal) were processed and embedded in paraffin by standard techniques.
Longitudinal sections of 5 mm thick were stained with H&E and examined
by a pathologist blinded to the experimental groups. Colitis scores of each
segment were assigned based on the extent and severity of inflammation,
ulceration, and hyperplasia of the mucosa. Severity scores for inflam-
mation were as follows: 0, normal (within normal limits); 1, mild (small,
focal, or widely separated, limited to lamina propria); 2, moderate (mul-
tifocal or locally extensive, extending to submucosa); 3, severe (transmural
inflammation with ulcers covering .20 crypts). Scores for ulceration were
as follows: 0, normal (no ulcers); 1, mild (one to two ulcers involving up to
a total of 20 crypts); 2, moderate (one to four ulcers involving a total of
20–40 crypts); 3, severe (more than four ulcers or .40 crypts). Mucosal
hyperplasia scores were assigned as follows: 0, normal (within normal
limits); 1, mild (crypts two to three times normal thickness, normal epi-
thelium); 2, moderate (crypts two to three times normal thickness, hyper-
chromatic epithelium, reduced goblet cells, scattered arborization); 3, severe
(crypts more than four times normal thickness, marked hyperchromasia,
few to no goblet cells, high mitotic index, frequent arborization). Scoring
for extent of lesions was as follows: 0, normal (0% involvement); 1, mild
(up to 30% involvement); 2, moderate (30–70% involvement); 3, severe
(.70% involvement). The individual scores from the four segments were
summed such that the maximum colitis score for a given animal is 48 and the
minimum score is 0. For immunohistochemistry, formalin-fixed paraffin-
embedded tissues were cut into 4-mm sections and slides were stained with
Abs against the macrophage marker F4/80 (Caltag Laboratories, Burlingame,
CA) and phospho-STAT1 (Cell Signaling T echnology, Beverly, MA), re-
spectiv ely.
Cytokine measurements
To measure the cytokine levels in colon tissue, a part of the colon was
homogenized mechanically in PBS containing 1% Nonidet P-40 and
a complete protease inhibitor mixture tablet (Roche Diagnositics, India-
napolis, IN). Mouse cytokines and chemokines in serum and colon homo-
genates were determined with Luminex (Bio-Rad, Hercules, CA) and
ELISA (R&D Systems, Minneapolis, MN) assays.
Real-time RT-PCR
Total RNA from colon tissue was isolated with TRIzol (Invitrogen,
Carlsbad, CA). First-strand cDNA was synthesized from 250 ng of RNA
using SuperScript III (Invitrogen). Real-time PCR for cyclooxygenase
(COX)-2 and IFN-g was performed using SYBR Green Master mix
(Invitrogen) on an ABI Prism 7500 real-time PCR system (Applied Bio-
systems, Foster City, CA). mRNA levels were determined by means of the
standard curve method. A standard sample was serially diluted and used
for constructing a standard curve. Simultaneous quantification of GAPDH
mRNA was used as an internal control.
In situ intestinal proliferation assay
The number of proliferating cells in intestinal epithelium was determined
using the immunoperoxidase staining protocol with the thymidine analog
BrdU as described earlier (29). In brief, 1 mg/ml BrdU in PBS was injected
i.p. Three hours laber later, colon tissue was collected, fixed in 10% neutral
buffered formalin, and embedded in paraffin. Immunohistochemistry was
performed using an in situ BrdU staining kit (BD Biosciences, San Jose,
CA). Tissues were counterstained with hematoxylin.
Western blotting
Tissue homogenates were lysed in lysis buffer (10 mM Tris-HCl, 150 mM
Nacl, 5 mM EDTA, 0.1% Nonidet P-40, 0.25% sodium deoxycholate,
supplemented with protease and phosphatase inhibitor cocktails; Roche
Diagnostics), and membranes were removed by centrifugation at 11,000 3
g. Before s eparation by SDS-PAGE, protein samples were de natured
with SDS plus 100 mM DTT and boiled for 5 min. Separated proteins
were transferred to polyvinylidene difluoride membranes and immu-
noblotted with primary Abs against phospho-STAT1, STAT1, rabbit
phospho-IkB, IkB (all from Cell Signaling Technology), and b-actin
(Sigma-Aldrich).
Recombinant IL-18 and IFN-g treatment
Recombinant IL-18 (MBL International, Woburn, MA) was injected i.p. at
a concentration of 0.5 mg/mouse on days 0, 2, and 4 and at 0.1 mg/mouse
on days 6 and 8 after DSS administration. Alternatively, Casp1
2/2
mice
were injected i.p. with recombinant mouse IFN-g (R&D Systems) at
a concentration of 200 IU/mouse on days 0, 2, 4, and 6 after DSS ad-
ministration.
Statistical analysis
Data are represented as mean 6 SE. Statistical significance was determined
by a Student t test or x
2
test. The p values ,0.05 were considered sta-
tistically significant.
Results
Increased susceptibility to colitis-associated colorectal tumor
formation in Nlrp3 inflammasome-deficient mice
Nlrp3-deficient mice were recently shown to be highly susceptible
to the induction of inflammation and tissue damage in the acute
DSS-induced colitis model (29). Similarly, Nlrp3-deficient mice
developed more severe symptoms of chronic colitis when mice
were administered multiple cycles of DSS (28, 29). To deter-
mine whether the increased and prolonged gut inflammation in
inflammasome-deficient mice led to increased tumorigenesis,
Nlrp3
2/2
, ASC
2/2
, and Casp1
2/2
mice were administered a single
dose of the DNA methylating agent AOM (10 mg/kg), followed
by repeated cycles of a 3% DSS solution (31). Twelve weeks after
AOM injection (Fig. 1A), the development of adenomatous polyps
and well-formed tumors in the colons of Nlrp3 inflammasome-
deficient mice was examined and compared with colons of treated
wild-type mice. Mice in the wild-type, Nlrp3
2/2
, ASC
2/2
,and
Casp1
2/2
groups developed tumors after AOM and DSS adminis-
tration, but tumor burdens were signicantly increased in Nlrp3
2/2
,
ASC
2/2
,andCasp1
2/2
mice over wild-type mice (Fig. 1B,1C).
Nevertheless, statistically significant differences in tumor size could
The Journal of Immunology 4913
not be observed (data not shown). Most tumors were located in the
distal area of the colon in all genotypes (Fig. 1D), although a fraction
of the tumors were found in the midcolon section (Fig. 1E). Notably,
the number of mice presenting with tumors in the midcolon region
increased from 40% of wild-type mice to .60% in the Nlrp3
2/2
and Casp1
2/2
cohorts (Fig. 1F). This may be due to the more severe
inflammation and extended tissue damage that DSS induced in
Nlrp3
2/2
and Casp1
2/2
mice (27–29).
Colons and ceca of representative tumor-bearing wild-type,
Nlrp3
2/2
, and Casp1
2/2
mice were sectioned and stained with
H&E to study mucosal dysplasia in more detail. Significantly
more dysplastic cells and hyperplastic areas, adenomatous polyps,
and well-formed tumors were visible in the distal colons of
Nlrp3
2/2
and Casp1
2/2
mice relative to those of wild-type mice
(Fig. 2A). Moreover, the number of mice presenting with dys-
plastic events was significantly higher in the Nlrp3
2/2
and
Casp1
2/2
cohorts (Fig. 2B). In all three genotypes, tumors were
mainly derived from dysplastic epithelial cells at the site of in-
flammation (Fig. 2C). The tumors appeared as sessile tubulo-
villous adenomas, and evidence for adenocarcinoma development
was not observed. Histopathological scoring for severity of in-
flammation, inflamed area, ulceration, and hyperplasia in the co-
lon and cecum was in agreement with significantly increased
disease progression in Nlrp3
2/2
and Casp1
2/2
mice (Fig. 2D). As
indicated before (Fig. 1D), most tumors were located in the distal
area of the colon, but occasionally tumors were found in the
midcolon area. Separate histopathological scorings for the distal
and midcolon regions were performed to determine whether spa-
tial differences in pathology could be observed. In both the distal
(Fig. 2E) and midcolon (Fig. 2F) regions, read-outs for in-
flammation severity, inflamed area, and hyperplasia were signifi-
cantly increased in Nlrp3
2/2
and Casp1
2/2
mice over wild-type
controls. Collectively, these results demonstrate that a functional
Nlrp3 inflammasome is critical for protection against colitis-
associated dysplasia and tumorigenesis in the gut. These obser-
vations are in agreement with a recent report showing increased
AOM/DSS-induced colon tumorigenesis in mice lacking Nlrp3 or
the inflammasome effectors ASC and caspase-1 (28).
Enhanced tumorigenesis in Nlrp3 inflammasome-deficient mice
is associated with deregulated IL-18 production and increased
macrophage infiltration
The Nlrp3 inflammasome is required for maturation and secretion
of the inflammatory cytokines IL-1b and IL-18 (32). To determine
whether the absence of a functional Nlrp3 inflammasome affects
local IL-1b and IL-18 production in the gut during early stages
of tumorigenesis, the levels of these cytokines were measured
in colon homogenates of Nlrp3
2/2
and Casp1
2/2
mice 5 d after
completion of the first DSS cycle (day 10 after AOM treatment).
IL-1b amounts in the colon homogenates of AOM/DSS-treated
wild-type, Nlrp3
2/2
, and Casp1
2/2
mice remained low and
barely rose above those of untreated animals (Fig. 3A and data not
shown). In contrast, significant levels of IL-18 were measured in
colon homogenates of AOM/DSS-treated wild-type mice (Fig.
3A). However, IL-18 levels in colon homogenates of Nlrp3
2/2
and
Casp1
2/2
mice were nearly 50% lower than those of treated wild-
type controls (Fig. 3A). Unlike IL-18, the levels of the cytokines
IL-6, IL-12, and TNF-a did not differ significantly from those
found in wild-type controls (data not shown), demonstrating the
specificity of these results. Moreover, we measured significantly
higher levels of the chemokines MIP-1a and eotaxin in colon
homogenates of Nlrp3
2/2
and Casp1
2/2
mice (Fig. 3B,3C),
suggesting that deregulated IL-18 production triggered an in-
creased recruitment of inflammatory cells in colons of Nlrp3
2/2
and Casp1
2/2
mice. In agreement, colon sections of the latter
genotypes contained significantly more F4/80-positive cells than
did wild-type colons (Fig. 3D), indicating a dramatically increased
infiltration of macrophages in colons of Nlrp3
2/2
and Casp1
2/2
mice. Macrophages exert a regulating role in the colorectal tumor
microenvironment through the production of a variety of tumori-
genic factors including COX-2, which promotes tumor develop-
ment through the synthesis of its enzymatic product PGE
2
(33).
Consistent with the increased macrophage infiltration in colons of
Nlrp3
2/2
and Casp1
2/2
mice, real-time PCR analysis demon-
strated significantly higher COX-2 mRNA levels in colon tissue of
Nlrp3
2/2
and Casp1
2/2
mice (Fig. 3E).
We next sought to determine the effect of deregulated IL-18
production, increased macrophage infiltration, and COX-2 pro-
duction on epithelial cell proliferation in the colons of AOM/DSS-
treated Nlrp3
2/2
and Casp1
2/2
mice. To this end, epithelial cell
proliferation was examined by BrdU staining at the early and late
time points of 10 d and 12 wk after AOM injection, respectively.
Interestingly, the number of proliferating cells located in dys-
plastic regions of Nlrp3
2/2
and Casp1
2/2
colons was significantly
higher than in tumor tissue of AOM/DSS-treated wild-type mice at
both analyzed time points (Fig. 4). In contrast, no significant
differences in proliferation were noted between the three geno-
types in regions of the normal mucosa (Fig. 4). AOM-induced
mutagenic events are likely to be critical for inducing neoplasia
in the context of the colonic microenvironment of Nlrp3
2/2
and
FIGURE 1. Nlrp3
2/2
, ASC
2/2
,andCasp1
2/2
mice are hypersusceptible
to colitis-associated colorectal tumorigenesis. A,WT(n =15),Nlrp3
2/2
(n =15),ASC
2/2
(n = 8), and Casp1
2/2
mice (n = 13) were administered
AOM on day 0 and were then given a 3% DSS solution during three 5-d
cycles as described in Materials and Methods. B, Twelve weeks after AOM
injection, mice were sacrificed to determine tumor development in the
colon. C–E, Total tumor numbers observed in whole colon (C), distal colon
(D), and middle colon (E) were determined. F, The percentage of mice
containing tumors in the midcolon section was calculated. Data represent
means 6 SE. pp , 0.05. WT, wild-type.
4914 THE Nlrp3 INFLAMMASOME CONTROLS COLORECTAL TUMORIGENESIS
Casp1
2/2
mice because DSS administration alone failed to induce
increased cell proliferation in colonic crypts of Nlrp3
2/2
and
Casp1
2/2
mice (29). Taken together, these results suggest a criti-
cal role for Nlrp3 inflammasome-mediated production of IL-18 in
protection against colitis-associated immune cell invasion and
neoplasia.
FIGURE 2. Histopathological examination of tumor
and colon tissue of WT, Nlrp3
2/2
, and Casp1
2/2
mice.
WT, Nlrp3
2/2
, and Casp1
2/2
mice (n = 10/genotype)
were injected with AOM and then received three cycles
of a 3% DSS solution as described in Materials and
Methods. Twelve weeks after AOM injection, colons
were collected and sections were stained with H&E
for histopathological analysis. A, Low magnification
(original magnification 320) scanning of distal colon
after H&E staining. B, Overall grading of dysplasia in
each genotype was performed as described in Materials
and Methods. C, Representative high magnification
images of H&E staining showing dysplasia in colon
tissue of WT (low-grade dysplasia), Nlrp3
2/2
(low-
grade dysplasia), and Casp1
2/2
(high-grade dysplasia)
mice. Original magnification 320. D–F, Semiquantitati v e
scoring of inflammation, hyperplasia, and inflamed area
in total (D), distal (E), and midcolon (F) sections. Data
represent means 6 SE. pp , 0.05. WT, wild-type.
FIGURE 3. Decreased production of IL-18 in
Nlrp3
2/2
and Casp1
2/2
colons is associated with
increased inflammation and induction of tumori-
genic factors. WT, Nlrp3
2/2
,andCasp1
2/2
mice
were injected with A O M followed by 3% DSS
treatment. A–C, Distal colons were collected at
day 15 after A OM injection, and homogenates were
used to determine IL-1b and IL-18 levels (A)andthe
concentrations of the chemotactic factors MIP-1a
(B)andeotaxin(C)byELISA.D, Colon sections
were simultaneously immunostained for the macro-
phage marker F4/80. Original magnification 310. E,
Real-time PCR analysis was performed on distal
colon homogenates collected at day 15 after AOM
injection to measure COX-2 expression. Data rep-
resent means 6 SE (n = 5/group). pp , 0.05. WT,
wild-type.
The Journal of Immunology 4915
IL-18 signaling downstream of the Nlrp3 inflammasome
confers protection against colitis-associated tumorigenesis
To further examine the role of IL-18 in protection against colitis-
associated dysplasia and tumor development, we characterized
colon inflammation and tumor development in il-18
2/2
mice that
were subjected to the AOM/DSS regimen described in Fig. 1A.In
agreement with an important role for IL-18 in protection against
colitis-associated tumorigenesis, colons of il-18
2/2
mice contained
significantly more tumors than did those of treated wild-type mice
(Fig. 5A,5B). To determine whether increased tumor formation in
il-18
2/2
mice could be linked to increased epithelial cell damage
and colon inflammation during the early stages of disease, we
examined phenotypic and histological signs of colitis and hyper-
plasia during acute colitis. To this end, wild-type and il-18
2/2
mice
were administered AOM followed by a 3% DSS solution during
5 d before parameters of colitis development and tumorigenesis
were analyzed. The Il-18
2/2
mice presented with aggravated co-
litis, as evidenced by higher body weight loss (Fig. 5C), severe
inflammation, hyperplasia, and more dysplastic cells (Fig. 5D).
Semiquantitative scoring of histological colon sections for in-
flammation, ulceration, affected area, and hyperplasia was con-
sistent with markedly increased disease development in il-18
2/2
mice relative to the group of wild-type mice (Fig. 5E).
As a complementary approach to the use of il-18
2/2
mice, we
studied whether recombinant IL-18 could reverse disease pro-
gression in AOM/DSS-treated Casp1
2/2
mice. Importantly, the
group of Casp1
2/2
mice that received recombinant IL-18 lost
significantly less weight compared with Casp1
2/2
mice that were
refused the recombinant cytokine (Fig. 5F). Moreover, IL-18 ad-
ministration provided protection against histological signs of in-
flammation and dysplasia (Fig. 5G). Consistently, semiquantitative
scoring of inflammation, ulceration, affected area, and hyper-
plasia on histological colon sections was indi cative of milder
disease in IL-18–treated Casp1
2/2
mice (Fig. 5H). These re sults
demonstrate that IL-18 signaling downstream of the Nlrp3 in-
flammasome confers protection against colitis-associated colorec-
tal tumorigenesis.
The Nlrp3 inflammasome activates the tumor suppressor
STAT1 in the colon via IL-18–mediated IFN-g production
Our results showed that impaired production of IL-18 down-
stream of the Nlrp3 inflamm asome contributes to aggravated
colitis-associated tumorigenesis (Figs. 3, 5). IL-18 was initiall y
described as the cytokine responsible for induction of IFN-g
production (22), and IFN-g was a ttributed potent antitumor ac-
tivity in a variety of experimental tumorigenesis models (34–38).
In agreement with the defective IL-18 production in colons of
AOM/DSS-treated Nlrp3
2/2
and Casp1
2/2
mice (Fig. 3A), we
found that IFN-g mRNA levels were dramatically lower in colon
homogenates of the latter genotypes relative to those of AOM/
DSS-treated wild-type mice (Fig. 6A). Diminished IFN- g pro-
duction was co nfirmed at the protein level by IFN-g–specific
ELISA (Fig. 6B). IFN-g–mediated antitumor signaling involves
activation of the transcription factor STAT1 (36). Notably,
stat1
2/2
mice are highly susceptible to tumorigenesis, classify-
ing STAT1 as a tumor suppressor (39, 40). To determine whether
decreased production of IL-18 and IFN- g in colons of AOM/
DSS-treated Casp1
2/2
mice affected STAT1 a ctivation levels,
phospho-specific STAT1 Abs were used to examine STAT1 ac-
tivation by Western blotting. STAT1 activation was a conse-
quence of AOM/DSS treatment, because basal STAT1 activation
levels in wild-type colons were significantly upregulated fol-
lowi ng AOM/DSS treatment (Fig. 6C). However, AOM/DSS-
induced STAT1 activation was dramatically reduced in colons
of Casp1
2/2
mice during early stages of tumorigenesis (day 15
after AOM treatment) (Fig. 6C). In contrast, phosphorylation of
the NF-kB inhibitor IkB wa s not affected (Fig. 6D ), demon-
strating the specificity of these results. Immunohistochemical
analysis of wild-type colons indicated increased phospho-STAT1
activation in epithelial cells and infiltrating immune cells upon
AOM/DSS treatment (Fig. 6E). Colons of AOM/DSS-treated
Casp1
2/2
mice contained significantly less cells stai ning posi-
tive f or phospho-STAT1 (Fig. 6E), suggesting that STAT1 sig-
naling in epithelial and immune cells may both contribute to
protection against tumorigenesis.
FIGURE 4. Increased hyperplastic cell proliferation in colons of AOM/DSS-treated Nlrp3
2/2
and Casp1
2/2
mice. WT, Nlrp3
2/2
, and Casp1
2/2
mice
were injected with AOM followed by administration of a 3% DSS solution as described in Materials and Methods. Mice were injected i.p. with BrdU either
at day 15 or 12 wk after AOM injection. Colon sections were immunostained to determine BrdU-positive cells. Original magnification 310. WT, wild-type.
4916 THE Nlrp3 INFLAMMASOME CONTROLS COLORECTAL TUMORIGENESIS
In agreement with an important role for IFN-g in inducing
STAT1 activation, STAT1 phosphorylation was restored by treat-
ing Casp1
2/2
mice with 200 IU recombinant IFN-g at days 5, 7,
9, and 11 after AOM treatment (Fig. 6F). Similarly, administration
of recombinant IL-18 restored phospho-STAT1 levels in AOM/
DSS-treated Casp1
2/2
mice to those observed in colons of AOM/
DSS-treated wild-type mice (Fig. 6G). Thus, IL-18– and IFN-g
mediated activation of the tumor suppressor STAT1 may play
a critical role in protection against colitis-associated tumorigene-
sis upon activation of the Nlrp3 inflammasome.
Discussion
Chronic inflammation is increasingly recognized as a critical risk
factor for the development of colorectal cancer (4). Members of the
NLR protein family are expressed on epithelial and professional
APCs residing in the colonic mucosa and lamina propria and play
key roles in regulating the immune response against commensal
microorganisms in the gut. Notably, defective activation of the
NLR member NOD1 has been reported to enhance inflammatory
cytokine production against commensal bacteria in the gut and
prime the colorectal mucosa for increased cell proliferation and
tumor formation during colitis in mice (25). Moreover, mutations
in the NLR protein NOD2 are linked with the development of
Crohn’s disease in humans (41, 42). It has been established that
Crohn’s disease patients are at increased risk of developing
sporadic colorectal cancer (43). In agreement, polymorphisms in
the gene encoding NOD2 have been associated with increased
susceptibility to gastrointestinal tumorigenesis (44). More recently,
mutations in the gene encoding Nlrp3 were linked with increased
susceptibility to Crohn’s disease in humans (26). Recent reports
from our and other groups demonstrated that DSS-induced colitis in
Nlrp3-deficient mice is associated with an increased destruction of
the epithelial barrier in the gut, inducing systemic dispersion of
colonic microflora and an exaggerated inflammatory response (27,
29). Nlrp3 plays a central role in activation of caspase-1 and se-
cretion of the proinflammatory cytokines IL-1b and IL-18 (30).
Caspase-1–deficient mice also were hypersensitive to DSS- and
2,4,6-trinitrobenzene sulfonic acid-induced colitis (27, 29), in-
dicating that Nlrp3 protects against colitis through the production of
caspase-1–dependent cytokines. Indeed, the phenotype of Casp1
2/2
mice was rescued by administration of recombinant IL-18 (27, 29).
Moreover, mice lacking the inflammasome inhibitor caspase-12
were resistant to acute colitis, although (paradoxically) they were
more susceptible to AOM/DSS-induced colorectal tumorigenesis
(27).
In this study, we showed that increased inflammatory responses
and destruction of the epithelial barrier led to enhanced dysplasia
and tumorigenesis in colons of AOM/DSS-treated Nlrp3
2/2
and
Casp1
2/2
mice. Our observations are in agreement with a recent
report showing that mice lacking Nlrp3, ASC, or caspase-1 were
FIGURE 5. IL-18 signaling downstream of
caspase-1 is critical for protection against
colitis-associated tumorigenesis. WT and Il-18
2/2
mice were administered AOM on day 0 and
were then given a 3% DSS solution during
three 5-d cycles as described in Materials
and Methods. A, Eleven weeks after AOM in-
jection, mice were sacrificed to determine tu-
mor development in the colon. B, Tumors
observed in the whole colon were counted. C,
Body weight change of WT and Il-18
2/2
mice
was monitored for 9 d after DSS administra-
tion. D, Representative images of inflamed and
hyperplastic areas in the distal colon at day 10
after DSS administration. H&E staining; orig-
inal magnification 310. E, Semiquantitative
histological scoring of inflammation, ulcera-
tion, and hyperplasia in whole colons of WT
and Il18
2/2
mice at day 10 after DSS admin-
istration. F, Casp1
2/2
mice were treated with
recombinant IL-18 on days 0, 2, 4, 6, and 8
after DSS administration. Body weight change
was monitored for 9 d. G, Representative
images of inflamed and hyperplastic areas in
the distal colon at day 10 after DSS administra-
tion. H&E staining; original magnification 310.
H, Semiquantitati v e scoring of inflammation,
ulceration, and hyperplasia in the whole colon at
day 10 after DSS treatment. Data represent
means 6 SE (n = 5/group). pp , 0.05. WT , wild-
type.
The Journal of Immunology 4917
hypersusceptible to AOM/DSS-induced colorectal tumor formation
(28). However, the mechanism by which the Nlrp3 inflammasome
confers protection against colitis-associated tumorigenesis remained
obscure (45). We demonstrated that IL-18 production was signi-
ficantly reduced in colons of AOM/DSS-treated Nlrp3
2/2
and
Casp1
2/2
mice, and, more importantly, that colons of AOM/DSS-
treated il-18
2/2
mice recapitulated the increased tumor burdens
seen in mice lacking Nlrp3 or caspase-1. These results suggested
a critical role for IL-18 production downstream of the Nlrp3
inflammasome in protection against colitis-associated neoplasia. In
agreement, administration of recombinant IL-18 markedly reduced
disease progression in A OM/DSS-treated Casp1
2/2
mice.
IL-18 was previously assigned an antitumor func tion in a variety
of exper imental tumor models (46–49). It was reported to inhibit
tumor growth and angiogenesis (50–52) and was associated with
repai r and restitution of ulcerated epithelium (53). We and others
showed that IL-18 is involved in repair of the epithelial layer of
the gut by maintaining proper levels of epithelial cell proliferat ion
during the acute stage of DSS-induced colitis (27, 29). DSS-
induced damage and erosion of the epithelial layer is repaired
by rapid proliferation of stem cells residing at the base of crypt s
(54). Intriguingly, while IL-18 promotes enterocyte proliferation
to repair chemically induced injury of colonic epithelium, we
showed in this study that it also inhibits hyper plasia during
chronic stages of colitis. This apparent discrepancy may be ex-
plained by differentia l roles of IL-18 during the acute and chronic
stages of colitis (53). Moreover, we only observed higher pro-
liferation rates in dysplastic regions of the colon epithelium of
AOM/DSS-treated Nlrp3
2/2
and Casp1
2/2
mice, but not in non-
tumor regions. These observations suggest that IL-18 exerts its
protective effect in two stages . First, during acute DSS-induced
colitis, it contributes to restoring epithelial barrier integrity by
induced controlled proliferation of stem cells at the cr ypt base
and turnover of damaged epithelial cells. This prevents systemic
dispersion of commensal microflora and the induct ion of exag-
gerat ed inflammatory responses. However, during remiss ion and
chronic stages of co litis, IL-18 inhibits epithelial cell proliferation
in neoplastic regio ns of the colon epithelium. This may be ach-
ieved, at least in part, through the induction of I FN-g production.
Indeed, IL-18 was originally identified as the “IFN-g–inducing
facto r” (22), and IFN-g has been described as a pleiotropic cy-
tokine with potent antitumor activity (34, 37). In this rega rd, we
demonstrated a markedly diminished production of IFN-g in
colons of AOM/DSS-treated Nlrp3
2/2
and Casp1
2/2
mice. No-
tably, IFN-g signaling was previously shown to confer protectio n
against experimental colitis (55). In agreement with a biphasic
role fo r Nlrp3-mediated IL-18 production in colitis-associated
tumorigenesis, a recent report described a biphasic role for
IFN-g during DSS-induced colitis with promotion of intestinal
epithelial cell proliferation at early stages and induction of anti-
proliferative responses at lat er stages (56). IFN-g mediates its
effect through IFN-gR, which is expressed on both normal and
malignant cells (57). Its biological effects are mediated by a
number of intracellular signaling pathways, the best characterized
of which is the JAK-STAT pathway. O nce IFN-gRisactivated,it
phosphorylates JAK1 and JAK2, which further induces phos-
phoryl ation and nuclear translocation of STAT1. We showed that
phosphorylated STAT1 levels were marked ly reduced in colons of
AOM/DSS-treated Casp1
2/2
mice, but they were restored upon
stimulation with either IFN- g or IL-18. These results indicate that
STAT1 signaling is affected in the absence of a functional Nlrp3
inflammasome. Once in the nucleus, STAT1 bi nds with g-acti-
vated sequences in IFN-g–responsive genes to induce transcrip-
tion of genes involved in cell proliferation, differentiatio n and cell
death (58, 59). Thus, IFN-g–mediated STAT1 activation down-
stream of IL-18 may play an important role in maintaining gut
homeostasis and inhibiting tumor development dur ing colit is.
In conclusion, we characterized the role and the mechanism by
which activation of the Nlrp3 inflammasome confers protection
FIGURE 6. The Nlrp3 inflammasome activates the tumor suppressor
STAT1 via IL-18-mediated IFN-g production. A and B,WT,Nlrp3
2/2
, and
Casp1
2/2
mice were administered AOM on day 0 and were then given
a 3% DSS solution for 5 d as described in Materials and Methods. IFN-g
production in colon tissue at day 15 after AOM injection was analyzed by
real-time PCR (A) and ELISA (B). Data represent means 6 SE (n =5/
group). pp , 0.05. C and D, Homogenates of the distal colon were pre-
pared 15 d after AOM injection and analyzed for total STAT1, phospho-
STAT1, total IkB, and phospho-IkB by Western blotting. b-actin was used
as a loading control. E, Colon tissue sections collected from control mice
and AOM/DSS-treated WT and Casp1
2/2
mice (at day 15 after AOM)
were immunostained for phospho-STAT1. Black arrows indicate phospho-
STAT1–positive epithelial cells; blue arrows indicate phospho-STAT1–
positive inflammatory cells. Original magnification 340. F, Casp1
2/2
mice were treated with IFN-g at 5, 7, 9, and 11 d after AOM adminis-
tration. On day 15 after AOM treatment, STAT1 activation in colon
homogenates was compared with that of AOM/DSS-treated WT and
Casp1
2/2
mice by Western blotting. G, Casp1
2/2
mice were treated with
IL-18 at 5, 7, 9, and 11 d after AOM treatment. On day 15 after AOM
treatment, STAT1 activation in colon homogenates was compared with that
of AOM/DSS-treated WT and Casp1
2/2
mice by Western blotting. WT,
wild-type.
4918 THE Nlrp3 INFLAMMASOME CONTROLS COLORECTAL TUMORIGENESIS
against the development of inflammation-associated colorectal tu-
morigenesis. We showed that Nlrp3 inflammasome-dependent IL-18
production prevents neoplastic events, possibly through the induction
of IFN-g production and STAT1 signaling. These results suggest
that strategies aimed at producing or delivering mature IL-18 in the
colon may prove beneficial in preventing colorectal tumor deve-
lopment in the context of chronic inflammation.
Acknowledgments
We thank Anthony Coyle, Ethan Grant, John Bertin (Millennium Pharma-
ceuticals, Cambridge, MA), Gabriel Nun
˜
ez (University of Michigan, Ann
Arbor, MI), and Richard Flavell (Yale University, New Haven, CT) for
a generous supply of mutant mice. We also thank Dorothy Bush (Veteri-
nary Pathology Core, St. Jude Children’s Research Hospital, Memphis,
TN) for technical assistance.
Disclosures
The authors have no financial conflicts of interest.
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4920 THE Nlrp3 INFLAMMASOME CONTROLS COLORECTAL TUMORIGENESIS
    • "The NLRP6 and NLRP12 inflammasomes have been linked to the maintenance of intestinal homeostasis, primarily in the context of chemical-induced colitis (Zaki et al. 2010; Elinav et al. 2011). Studies have observed that NLRP6 and NLRP12 suppress colon inflammation through inhibition of NF-κB signaling (Zaki et al. 2011; Allen et al. 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Inflammasomes are macromolecular cytoplasmic complexes that act as signaling platforms for the activation of inflammatory caspases. Their activation triggers the processing and secretion of the pro-inflammatory cytokines IL-1β and IL-18, as well as the induction of a specialized form of inflammatory cell death termed pyroptosis. Here, we review the mechanisms of inflammasome activation triggered by the intracellular pathogen Salmonella enterica serovar Typhimurium. We highlight the different inflammasome subfamilies utilized by macrophages, neutrophils, dendritic cells, and intestinal epithelial cells response to a Salmonella infection as well as the Salmonella ligands that trigger each inflammasome’s formation. We also discuss the evasion strategies utilized by Salmonella to avoid inflammasome detection. Overall, inflammasomes play a key and multilayered role at distinct stages of host cell defense against Salmonella infection.
    Full-text · Chapter · Jul 2016 · Journal of leukocyte biology
    • "The contributions of ROS and the inflammasomes that are induced by mitochondria in cancer cells are controversial; they can positively affect cell-autonomous death pathways and anticancer immunosurveillance, but they may also stimulate autocrine or paracrine processes that favor carcinogenic inflammation, tumor growth, metastasis and angiogenesis (Zitvogel et al., 2012). A protective role for NLRP3 has been described in hepatocellular carcinoma (Wei et al., 2014); NLRP3 and caspase-1 null mice are more susceptible to azoxymethane/dextran sulfate sodiuminduced carcinogenesis (Allen et al., 2010; Zaki et al., 2010). Based on this evidence, the discovery of NLRC4 as a downstream transcriptional target of p53 constituted promising evidence for the anti-tumorigenic functions of NLR (Sadasivam et al., 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Over the last few decades, many different groups have been engaged in studies of new roles for mitochondria, particularly the coupling of alterations in the redox pathway with the inflammatory responses involved in different diseases, including Alzheimer's disease, Parkinson's disease, atherosclerosis, cerebral cavernous malformations, cystic fibrosis and cancer. Mitochondrial dysfunction is important in these pathological conditions, suggesting a pivotal role for mitochondria in the coordination of pro-inflammatory signaling from the cytosol and signaling from other subcellular organelles. In this regard, mitochondrial reactive oxygen species are emerging as perfect liaisons that can trigger the assembly and successive activation of large caspase-1-activating complexes known as inflammasomes. This review offers a glimpse into the mechanisms by which inflammasomes are activated by mitochondrial mechanisms, including reactive oxygen species production and mitochondrial Ca2+ uptake, and the roles they can play in several inflammatory pathologies.
    Article · Jun 2016
    • "However, in diseases such as colitis and fungal infection, NLRP3 was shown to have beneficial roles [18,[48][49][50][51][52]. In colitis, the absence of NLRP3 reduced the levels of IL-18, which was shown to be critically involved in the maintenance of intestinal homeostasis [50].With the fungal infection, the explanation for more-severe lesions in the absence of NLRP3 was attributed to the impaired IL-1b response [18, [48][49][50][51][52][53]. Our study is the first, to our knowledge, to evaluate any role for NLRP3 during an in vivo infection with HSV. "
    [Show abstract] [Hide abstract] ABSTRACT: Herpes simplex 1 infection of the eye can cause blindness with lesions in the corneal stroma largely attributable to inflammatory events that include components of both adaptive and innate immunity. Several innate immune responses are triggered by herpes simplex 1, but it is unclear how such innate events relate to the subsequent development of stromal keratitis. In this study, we compared the outcome of herpes simplex 1 ocular infection in mice unable to express NLRP3 because of gene knockout (NLRP3(-/-)) to that of wild-type mice. The NLRP3(-/-) mice developed more-severe and earlier stromal keratitis lesions and had higher angiogenesis scores than did infected wild-type animals. In addition, NLRP3(-/-) mice generated an increased early immune response with heightened chemokines and cytokines, including interleukin-1β and interleukin-18, and elevated recruitment of neutrophils. Increased numbers of CD4(+) T cells were seen at later stages of the disease in NLRP3(-/-) animals. Reduction in neutrophils prevented early onset of the disease in NLRP3(-/-) animals and lowered levels of bioactive interleukin-1β but did not lower bioactive interleukin-18. In conclusion, our results indicate that NLRP3 has a regulatory and beneficial role in herpetic stromal keratitis pathogenesis.
    Article · Oct 2015
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