NLRP6 Inflammasome Regulates
Colonic Microbial Ecology
and Risk for Colitis
Eran Elinav,1,8Till Strowig,1,8Andrew L. Kau,4,5Jorge Henao-Mejia,1Christoph A. Thaiss,1Carmen J. Booth,2
David R. Peaper,3John Bertin,6Stephanie C. Eisenbarth,1,3Jeffrey I. Gordon,4and Richard A. Flavell1,7,*
1Department of Immunobiology
2Section of Comparative Medicine
3Department of Laboratory Medicine
Yale University School of Medicine, New Haven, CT 06520, USA
4Center for Genome Sciences and Systems Biology
5Division of Allergy and Immunology, Department of Internal Medicine
Washington University School of Medicine, Saint Louis, MO 63108, USA
6Pattern Recognition Receptor Discovery Performance Unit, GlaxoSmithKline, Collegeville, PA 19426, USA
7Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
8These authors contributed equally to this work
Inflammasomes are multiprotein complexes that
function as sensors of endogenous or exogenous
damage-associated molecular patterns. Here, we
show that deficiency of NLRP6 in mouse colonic
epithelial cells results in reduced IL-18 levels and
altered fecal microbiota characterized by expanded
representation of the bacterial phyla Bacteroidetes
(Prevotellaceae) and TM7. NLRP6 inflammasome-
deficient mice were characterized by spontaneous
intestinal hyperplasia, inflammatory cell recruitment,
and exacerbation of chemical colitis induced by
exposure to dextran sodium sulfate (DSS). Cross-
fostering and cohousing experiments revealed that
the colitogenic activity of this microbiota is transfer-
able to neonatal or adult wild-type mice, leading to
exacerbation of DSS colitis via induction of the
cytokine, CCL5. Antibiotic treatment and electron
microscopy studies further supported the role of
Prevotellaceae as a key representative of this micro-
biota-associated phenotype. Altogether, perturba-
tions in this inflammasome pathway, including
NLRP6, ASC, caspase-1, and IL-18, may constitute
a predisposing or initiating event in some cases of
The distal intestine of humans contains tens of trillions of
Eukarya and their viruses. The vast repertoire of microbial genes
myriad functions that benefit the host (Qin et al., 2010). The
mucosal immune system coevolves with the microbiota begin-
ning at birth, acquiring the capacity to tolerate components of
the microbial community while maintaining the capacity to
respond to invading pathogens. The gut epithelium and its over-
lying mucus provide a physical barrier. Epithelial cell lineages,
notably the Paneth cell, sense bacterial products through recep-
tors for microbe-associated molecular patterns (MAMPs), result-
ing in regulated production of bactericidal molecules (Vaishnava
et al., 2008). Mononuclear phagocytes continuously survey
and the initiation of immune responses (Macpherson and Uhr,
2004; Niess et al., 2005; Rescigno et al., 2001).
Several families of innate receptors expressed by hematopoi-
etic and nonhematopoietic cells are involved in recognition
lectin receptors (Geijtenbeek et al., 2004; Janeway and Medzhi-
proteins, including NLRP1, NLRP3, and NLRC4, which function
ciated molecular patterns (Schroder and Tschopp, 2010). Upon
sensing the relevant signal, they assemble, typically together
with the adaptor protein, apoptosis-associated speck-like
protein (ASC), into a multiprotein complex that governs cas-
matory cytokines, including pro-IL-1b and pro-IL-18 (Agostini
et al., 2004; Martinon et al., 2002).
Several other members of the NLR family, including NLRP6
and NLRP12, possess the structural motifs of molecular sensors
and are recruited to the ‘‘specks’’ formed in the cytosol by ASC
oligomerization, leading to procaspase-1 activation (Grenier
Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc. 745
et al., 2002; Wang et al., 2002). However, the triggers and func-
tion of NLRP12 are only now being revealed (Arthur et al., 2010),
and those of NLRP6 remain unknown. In this study, we describe
a mechanism for how the immune system regulates colonic mi-
crobiota via an inflammasome that requires NLRP6, ASC, and
caspase-1 and leads to the cleavage of pro-IL-18. In mice that
are deficient in NLRP6, ASC, caspase-1, or IL-18, gut microbial
ecology is altered, with prominent changes in the representation
of members of several bacterial phyla. Strikingly, this altered mi-
crobiota is associated with a colitogenic phenotype that is trans-
missible to cohoused wild-type mice, both early in postnatal life
and during adulthood.
ASC-Deficient Mice Develop Severe DSS Colitis
that Is Transferable to Cohoused WT Mice
To characterize possible links between inflammasome function
and homeostasis achieved between the innate immune system
and the gut microbiota, we studied mice that are deficient
in ASC. A more severe colitis developed after dextran sodium
sulfate (DSS) administration to single-housed ASC?/?mice
(National Cancer Institute, NCI) (Figure 1A and data not shown).
Remarkably, cohousing of adult ASC?/?mice with age-matched
6 10 128420
Mass change (%)
Mass change (%)
Mass change (%)
WT WT (ASC-/-) ASC-/- (WT)
Figure 1. The Increased Severity of Colitis in ASC-Deficient Mice Is Transmissible to Cohoused Wild-Type Mice
(A and B)Toinduce colitis, micewere given 2%DSSintheirdrinking waterfor 7days. (A) Weight lossof ASC?/?mice and separately housed wild-type (WT) mice.
(B) ASC?/?mice and WT mice were cohoused for 4 weeks, after which DSS colitis was induced.
(C–F) Weight loss (C), colonoscopy severity score at day 7 (D), and survival (F) after induction of DSS colitis of WT mice that were cohoused with (i) in-house WT
mice bred for several generations in our vivarium (IH-WT) or (ii) ASC?/?mice (designated WT(IH-WT) and WT(ASC?/?), respectively). (E) Representative images
taken during colonoscopy of mice at day 7.
(Gand H)RepresentativeH&E-stained sectionsofcolons fromWT(IH-WT),WT(ASC?/?),and ASC?/?(WT)micesampled onday 6(G) and day12(H)after thestart
of DSS exposure. Epithelial ulceration (arrowheads), severe edema/inflammation (asterisk) with large lymphoid nodules (L), retention/regeneration of crypts
(arrows), and evidence of re-epithelialization/repair of the epithelium (box).
Scale bars, 500 mm. Data are representative for three independent experiments. Error bars represent the SEM of samples within a group. *p < 0.05 by one-way
ANOVA. For related data, see Figures S1A–S1D.
746 Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc.
WT mice for 4 weeks prior to induction of DSS colitis resulted in
development of comparably severe DSS-induced colitis in
ASC?/?as well as cohoused WT mice (the latter are designated
‘‘WT(ASC?/?)’’ in Figure 1B).
To assess the possibility that differences in colitis severity
observed between groups of single-housed ASC?/?and WT
mice were indeed driven by differences in their intestinal micro-
biota, WT mice were cohoused for 4 weeks with either ASC?/?
mice (WT(ASC?/?)) or WT mice that had been bred in our
vivarium for more than ten generations (in-house mice (IH-WT),
WT(IH-WT)). The severity of DSS-induced colitis was similar
among NCI-WT (data not shown), IH-WT, and WT(IH-WT) as
well as IH-WT(WT) as judged by weight loss (Figure 1C), colitis
severity score (defined by colonoscopy) (Figures 1D and 1E),
and survival (Figure 1F). In contrast, WT(ASC?/?) and ASC?/?
mice were characterized by an equally increased severity of
disease compared to these other groups at both early and late
stages (Figures 1C–1H and Figures S1A–S1D available online).
To further establish the role of the intestinal microbiota, we
performed cross-fostering experiments. Newborn ASC?/?mice
cross-fostered (CF) at birth with in-house WT mothers (CF-
ASC?/?) exhibited milder colitis compared to noncross-fostered
ASC?/?mice (Figures 2A and 2B). In contrast, newborn WT mice
Mass change (%)
Mass change (%)
Mass change (%)
WT WT (ASC-/-) WT (CF-ASC-/-)
Figure 2. Maternal Transmission of an Exacer-
bated DSS Colitis Phenotype
(A–F) Newborn ASC?/?and WT mice were swapped
followed by induction of acute DSS colitis at 8 weeks of
age. Body weight and colonoscopy severity score were
measured in ASC?/?mice and ASC?/?mice cross-
fostered withWT mothers (CF-ASC?/?)(A and B);WT mice
and WT mice cross-fostered with ASC?/?
(CF-WT) (C and D); WT mice cohoused with ASC?/?or
cross-fostered ASC?/?mice for 4 weeks (E and F). Data
are representative of three independent experiments.
Error bars represent the SEM of samples within a group.
*p < 0.05 by one-way ANOVA. Figures S1E–S1G contain
cross-fostered with ASC?/?mothers (CF-WT)
developed severe colitis in comparison to non-
cross-fostered WT mice (Figures 2C and 2D).
Moreover, CF-ASC?/?mice were no longer
able to transmit enhanced colitis to cohoused
WT mice (Figures 2E and 2F).
mice and subsequent housing with naive WT
mice resulted in a gradual partial reduction in
colitis severity compared to WT(ASC?/?) mice
that were not exposed to a WT microbiota
(Figures S1E–S1G). Together, these results
demonstrate that the ASC?/?microbiota is
is sustainable in recipient mice for prolonged
periods of time. Nonetheless, exposure of an
established transferred ASC?/?-derived micro-
biota in a WT mouse to WT microbiota amelio-
rates its colitogenic potential, suggesting that the latter commu-
nity can displace the former and diminish its disease-promoting
properties in WT mice.
Culture-independent methods were subsequently employed to
variableregion2 (V2) of bacterial 16SrRNAgenes present infecal
samples collected from ASC?/?and WT mice just prior to and
jected to multiplex pyrosequencing, and the resulting chimera-
checked and filtered data sets were compared using UniFrac
(mean of 3524 ± 1023 [SD] 16S rRNA reads/sample; see Experi-
in fecal bacterial phylogenetic architecture in WT versus ASC?/?
mice. Moreover, after 4 weeks of cohousing, the fecal bacterial
communities of WT(ASC?/?) mice clustered together with
rialcomponent of the fecal microbiota ofthese cohousedASC?/?
mice was similar to ASC?/?mice that never had been cohoused.
NLRP6-Deficiency Produces a Microbiota-Mediated
Phenotype that Resembles that of ASC Deficiency
To assess whether ASC’s function as adaptor protein for inflam-
masome formation is linked to the changes in gut bacterial
Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc. 747
community structure and function observed, we cohoused WT
mice with caspase-1?/?mice, and these too exhibited more
severe DSS-induced colitis compared to single-housed WT
mice (Figures S2A–S2E). Similar to WT mice cohoused with
ASC?/?mice, WT mice cohoused with caspase-1?/?mice
evolved their intestinal bacterial communities to a phylogenetic
configuration that was very similar to that of their caspase-1?/?
cagemates (Figure S2F). These results point to the involvement
of an inflammasome in this phenotype.
We next sought to identify the NLR(s) upstream of ASC and
caspase-1 leading to the phenotype. qRT-PCR analysis of 24
tissues in WT mice revealed that NLRP6, which forms an ASC-
dependent inflammasome (Grenier et al., 2002), is most highly
expressed in the gastrointestinal tract and at lower levels in
lung, kidney, and liver (Figure 4A). Further, we isolated RNA
prepared from colonic epithelium and sorted colonic CD45+
hematopoietic cells and found that ASC and caspase-1 are
110 100 0.10.01
Favors KO and
Cohousing - +
Cohousing - +
Cohousing - +
Cohousing - +
Figure 3. Bacterial 16S rRNA-Based Anal-
ysis of the Fecal Microbiota of WT and
NLRP6 Inflammasome-Deficient Mice
(A–D) Unweighted UniFrac PCoA of fecal micro-
biota harvested from WT mice single-housed or
cohoused with ASC?/?(A), IL-18?/?(B), NLRP6?/?
(C), or all (D) mice. Samples from mice shown in (A)
and (C) were taken just prior to cohousing and
28 days later. Dashed line illustrates separation of
samples along PC1.
(E) Distribution of family-level phylotypes in ASC-,
IL-18-, NLRP6-deficient, and cohoused WT mice,
compared to single-housed WT mice. The hori-
zontal axis shows the fold representation (defined
as the ratio of the percentage of samples with
genera present in knockout or cohoused mice
in single-housed WT mice; the right denotes taxa
whose representation is greater in knockout or
cohoused WT mice. The origin represents equiv-
alent recovery of taxa in both groups. The vertical
axis shows the calculated p value for each taxa as
defined by G test. Open diamonds represent taxa
that were found only in KO/cohoused WT or
single-housed WT mice but where recovery was
assumed to be 1 to calculate fold representation.
(F) Unweighted UniFrac PCoA demonstrating
presence or absence of TM7 and Prevotellaceae
in each sample. Dashed lines show separation
of single-housed WT and cohoused WT and
knockout mice on PC1. PC2 in panels (D) and (F)
shows separation of communities based on
host genotype/cohousing. For additional data
related to the transmission of fecal microbiota in
inflammasome deficient mice, see Figure S2.
highly expressed in both compartments.
NLRP6 expression, in contrast, was
essentially limited to the epithelial com-
partment (Figure 4B). Indeed, in bone
marrow transfer experiments, NLRP6
was almost undetectable in NLRP6?/?
mice (Figures S3A and S3B) receiving
WT bone marrow (Figure 4C). Follow-up immunoprecipitation
(Figure 4D) and immunofluorescence assays (Figures 4E and
4F) both showed that NLRP6 protein is expressed in primary
speckled cytoplasmic aggregates, whereas it was absent in
WT and NLRP6?/?mice were then single housed or cohoused
for 4 weeks, followed by exposure to DSS. Single-housed
NLRP6?/?mice developed more severe colitis compared
to single-housed WT mice (Figures 4G–4J). The more severe
colitis phenotype was transferable to cohoused WT mice
(WT(NLRP6?/?)) (Figures 4G–4J and Figures S3C–S3G). 16S
rRNA analysis of fecal bacterial communities demonstrated
a clear difference in the bacterial community structure between
single-housed adult WT mice versus age-matched WT mice
cohoused for 4 weeks with NLRP6-deficient mice (Figure 3C).
Fecal bacterial communities of WT mice clustered together
748 Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc.
biota in turn was similar to NLRP6?/?mice that never had been
cohoused (Figure 3C).
To ascertain the specificity of this phenotype, we cohoused
WT mice with mice that lacked other NLR family members and
inflammasome-forming protein AIM2, all shown by qRT-PCR
analysis to be expressed in the colon (Figure S4A) (Kufer and
Sansonetti, 2011; Schroder and Tschopp, 2010). Adult, conven-
obtained from the same source as NLRP6?/?mice (Millenium,
Mass change (%)
Figure 4. NLRP6-Deficient Mice Harbor a Transmissible Colitogenic Gut Microbiota
(A and B) (A) Analysis of NLRP6 expression in various organs and (B) in colonic epithelial and hematopoietic (CD45+) cells. The purity of the sorted populations in
(B) was analyzed using vil1 and ptprc as markers for epithelial and hematopoietic cells, respectively.
(C) Bone marrow chimeras were generated usingWT and NLRP6?/?mice as host and bone marrow donor. NLRP6 expressionin the colon wasanalyzed 8 weeks
after bone marrow transplantation.
(D) Analysis of NLRP6 protein expression was performed by immunoprecipitation using an NLRP6 antibody and lysates of primary colonic epithelial cells isolated
from WT and NLRP6?/?mice.
(E and F) Representative confocal images of colonic sections analyzed for expression of NLRP6 (red) and counterstained with DAPI. (E) 403, (F) 1003. White
dotted lines were drawn to illustrate the epithelial cell boundaries.
(G–J) Acute DSS colitis was induced in single-housed WT mice, in WT mice cohoused for 4 weeks with NLRP6?/?mice (WT(NLRP6?/?), the corresponding
single-housed versus cohoused WT and NLRP6?/?mice. (J) Representative H&E-stained sections of colons on day 7 after initiation of DSS exposure.
Edema/inflammation (asterisks), ulceration (arrowheads), and loss of crypts (arrow). Scale bars, 500 mm. Data are representative of three independent experi-
ments. Error bars represent the SEM of samples within a group. *p < 0.05 by one-way ANOVA. Related data are in Figure S3 and Table S1.
Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc. 749
NLRP3?/?, NLRC4?/?, NLRP12?/?), generated in our own labo-
ratory (NLRP10?/?), or obtained from other laboratories
(AIM2?/?, K Fitzgerald, U. Massachusetts). NLRP3?/?mice
cohoused with WT mice for 4 weeks featured attenuated colitis
as compared to their WT cagemates and mild transferability of
colitis, suggesting that NLRP3’s major effect in this system is
negative regulation of the inflammatory process itself (data
not shown). Importantly, none of the other above mentioned
mouse strains transferred microbiota with increased colitogenic
properties to WT mice upon cohousing (Figures S4B–S4I). Like-
wise, 16S rRNA analysis of these strains revealed a distinct
configuration of their microbiota population as compared to
NLRP6 inflammasome-deficient mice (Table S1). Together,
these findings indicate that NLRP6 forms an intestinal epithelial
inflammasome that regulates functional properties of the micro-
biota and that loss of NLRP6 and the known inflammasome
constituents, ASC and caspase-1, leads to the specific develop-
ment of a transmissible, more colitogenic microbiota.
Evidence that NLRP6 Affects the Gut Microbiota
Activation of inflammasomes results in multiple downstream
effects, including proteolytic cleavage of pro-IL-1b and pro-IL-
18 to their active forms (Schroder and Tschopp, 2010). To test
whether the effect of NLRP6 deficiency is mediated via IL-1b
or IL-18 deficiency, we cohoused adult WT mice with either
IL-1b?/?(Figure 5A) or IL-1R?/?mice (Figures S4J and S4K).
Cohousing WT mice with these strains did not result in any
significant changes in the severity of DSS colitis compared to
single-housed WT mice, excluding a major contribution of the
IL-1 axis. In contrast, IL-18?/?mice and, more importantly, WT
mice cohoused with them exhibited a significant exacerbation
of colitis severity, compared to single-housed WT mice (Figures
In the steady state, single-housed NLRP6?/?mice had signif-
icantly reduced serum levels of IL-18 compared to their WT
counterparts and reduced production of this cytokine in their
colonic explants (Figures 5G and 5H). To study the relative
contribution of hematopoietic and nonhematopoietic NLRP6
deficiency to this reduction in active IL-18, we measured IL-18
protein levels in colonic explants prepared from chimeric mice
that had received bone marrow transplants from NLRP6?/?or
WT donors. Significantly lower IL-18 protein levels were noted
only in explants prepared from mice with NLRP6 deficiency in
the nonhematopoietic compartment (Figure 5I). This result indi-
cates that NLRP6 expressed in a nonhematopoietic component
of the colon, likely the epithelium, is a major contributor to
production of active IL-18. Furthermore, in contrast to WT
mice, NLRP6?/?mice failed to significantly upregulate IL-18 in
the serum and in tissue explants following induction of DSS
colitis (Figure 5J and data not shown).
To study whether IL-18 production by nonhematopoietic cells
is the major contributor to the microbiota-associated enhanced
colitogenic phenotype, we performed a bone marrow transfer
experiment using IL-18?/?and WT mice as both recipients and
donors. Indeed, mice that were deficient in IL-18 in the nonhe-
matopoietic compartment exhibited more severe disease
compared to mice that were sufficient for IL-18 in the nonhema-
topoietic compartment (Figures 5K and 5L). Bacterial 16S rDNA
studies demonstrated that the fecal microbiota of WT mice
as seen in the PC2 axis in the PCoA plot of unweighted UniFrac
distances, the fecal microbiota of ASC?/?and NLRP6?/?mice
were distinct from IL-18?/?mice, possibly reflecting the exis-
tence of additional NLRP6 inflammasome-mediated IL-18-inde-
pendent mechanisms of microflora regulation (Figure 3D).
Together, these results led us to conclude that the decrease in
colonic epithelial IL-18 production in mice that are deficient in
components of the NLRP6 inflammasome is critically involved
in the enhanced colitogenic properties of the microbiota.
The Gut Microbiota from NLRP6 Inflammasome-
Deficient Mice Induces CCL5 Production and Immune
Cell Recruitment,Leading toSpontaneous Inflammation
We next examined the intestines of untreated ASC?/?and
NLRP6?/?mice for signs of spontaneous pathological changes.
The colons, terminal ileums, and Peyer’s patches of ASC?/?and
NLRP6?/?mice exhibited colonic crypt hyperplasia, changes in
crypt-to-villus ratios in the terminal ileum, and enlargement of
Peyer’s patches with formation of germinal centers (Figure 6A
and Figures S5A and S5B). NLRP6 inflammasome-deficient
mice also had significantly elevated serum IgG2c and IgA
levels, as did cohoused WT mice (Figures S5C–S5F). In addition,
we recovered significantly more CD45+cells from colons of
NLRP6?/?mice compared to WT controls (Figure 6B). These
results prompted us to investigate downstream effector mecha-
nisms by which the altered microbiota could induce this immune
cell infiltration. Multiplex analysis of cytokine and chemokine
production by tissue explants (Figure S5G), followed by valida-
tion at the RNA (Figure 6C) and protein levels (Figure 6D),
indicated that CCL5 levels were significantly elevated in single-
caged untreated ASC?/?, NLRP6?/?, and IL-18?/?compared
to WT mice. Furthermore, CCL5 mRNA upregulation was found
to originate from epithelial cells (Figure 6E). Moreover, CCL5
levels were induced in WT mice upon cohousing (Figures 6F
and 6G), showing that this property was specified by the micro-
biota and not the mutated inflammasome per se. Notably, in the
representation of immune subsets with the exception of slight
reduction in gd TCR+lymphocytes, indicating that CCL5 is not
generally required for immune cell recruitment to the colon
To test the role of CCL5 in mediating the enhanced colitogenic
properties of the NLRP6?/?mouse microbiota, we cohoused
WT or CCL5?/?mice with NLRP6?/?mice for 4 weeks. We
subsequently induced DSS colitis and found comparable
colitis severity between single-housed WT and CCL5?/?mice
(Figures 6H and 6I). However, upon cohousing, WT(NLRP6?/?)
mice had significantly worse DSS-induced colitis compared
to CCL5?/?(NLRP6?/?) mice, despite comparable acquisition
of the NLRP6?/?colitogenic flora (Figure S5I). These findings
support the notion that CCL5 upregulation in response to the
altered microbiota is responsible for the exacerbation of colitis
that occurs in WT mice cohoused with NLRP6 inflammasome-
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Identification of Bacterial Phylotypes that Are Markedly
Expanded in Both NLRP6 Inflammasome-Deficient Mice
and in Cohoused WT Mice
To identify whether increased colitis severity is driven by bacte-
rial components, we first treated ASC?/?mice with a combina-
tion of antibiotics known to reduce the proportional representa-
tion of a broad range of bacterial phylotypes in the gut (Suzuki
et al., 2004; Rakoff-Nahoum et al., 2004). Antibiotic therapy
reduced the severity of DSS colitis in ASC?/?mice to WT
levels (Figures S6A and S6B). To exclude a possible role for
Mass change (%)
6 10 128420
Mass change (%)
Mass change (%)
Inflamed colon area (%)
Pathological colitis severity
Figure 5. Processing of IL-18 by NLRP6 Inflammasome Suppresses Colitogenic Microbiota
(A–C) WT mice were cohoused with IL-1b?/?mice or IL-18?/?mice for 4 weeks, and colitis was subsequently induced with DSS. Comparison of weight loss (A) in
single-housed WT mice and in WT mice previously cohoused with IL-1b?/?mice (WT(IL-1b?/?)). Weight loss (B) and colonoscopy severity score at day 7 (C) for
single-housed WT mice and WT mice previously cohoused with IL-18?/?mice (WT(IL-18?/?)).
(D–F) Representative H&E-stained sections (D) and pathologic quantitation of disease severity (E and F) of colons from single-housed WT mice and WT mice
cohoused with IL18?/?mice sampled 6 days after the start of DSS administration. Scale bars, 500 mm.
(G and H) IL-18 levels measured in sera (G) and colon explants (H) obtained from WT and NLRP6-deficient mice without treatment.
(I) Bone marrow chimeras were generated using both WT and NLRP6?/?mice as host and bone marrow donor. IL-18 production by colon explants was analyzed
8 weeks after bone marrow transplantation.
(J) IL-18 concentrations in the serum 5 days after induction of DSS colitis.
(KandL)Bonemarrow chimerasweregeneratedusingWTandIL-18?/?miceashostandbonemarrow donor.Weight(K)andcolonoscopyseverityscores atday
7 (L) of mice with acute DSS colitis are shown.
by one-way ANOVA. Related data are presented in Figure S4.
Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc. 751
herpesviruses, fungi, and parasites, single-housed WT and
ASC?/?mice were treated for 3 weeks with oral gancyclovir,
amphotericin, or albendazole and praziquantel, respectively.
None of these treatments altered the severity of colitis in ASC-
deficient mice (Figures S6C–S6E). Furthermore, fecal tests for
rotavirus, lymphocytic choriomeningitis virus, K87, murine cyto-
megalovirus, mouse hepatitis virus, mouse parvovirus, reovirus,
and Theiler’s murine encephalomyelitis virus were all negative,
WT (NLRP6-/-) WT
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Mass change (%)
Figure 6. Microbiota Induction of CCL5
(A) Representative H&E-stained sections of the colon, terminal ileum, and Peyer’s patches from WT, ASC?/?, and NLRP6?/?mice not exposed to DSS. Mucosal
hyperplasia in the colon (double arrows), increased crypt to villus ratio in the terminal ileum (asterisks), and enlargement of Peyer’s patches with formation of
germinal centers (arrowheads). Scale bars, 500 mm.
(B) Enumeration of subsets of hematopoietic cells harvested from the lamina propria of WT and NLRP6?/?mice.
(C and D) Analysis of CCL5 colonic mRNA expression (C) and protein expression in colonic explants (D) in WT, ASC?/?, NLRP6?/?, and IL-18?/?mice.
(E) CCL5 expression in epithelial cells from the colons of WT and NLRP6?/?mice.
(F and G) Analysis of CCL5 colonic mRNA expression (F) and protein expression in colonic explants (G) in single-housed WT mice and WT mice cohoused with
(H and I) WT and CCL5?/?mice were either single-housed or cohoused for 4 weeks with NLRP6?/?mice followed by exposure to DSS. Weight loss (H) and
colonoscopy severity score at day 7 (I) of mice after induction of acute DSS colitis.
SEM of samples within a group. *p < 0.05 by one-way ANOVA. Additional cytokine and chemokine analyses are presented in Figure S5.
752 Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc.
and there was no histological evidence of inclusion bodies,
which are characteristic of virally infected colonic epithelial cells
(data not shown). Together, these results pointed to bacterial
components as being responsible for the transferrable colitis
phenotype in NLRP6 inflammasome-deficient mice.
to (ii) ASC?/?and NLRP6?/?, and caspase-1?/?, and IL-18?/?,
and all types of cohoused WT mice (all untreated with DSS).
Nine genera belonging to four phyla (Firmicutes, Bacteroidetes,
Proteobacteria, and TM7) satisfied our requirement of having
significant differences in their representation in the fecal
that is most significantly associated with the fecal microbiota
of ASC?/?, NLRP6?/?, caspase-1?/?, IL-18?/?, and cohoused
WT mice was a member of the family Prevotellaceae in the
phylum Bacteroidetes. Beyond this unnamed genus in the
Prevotellaceae, the next two most discriminatory genus-level
taxa belonged to the phylum TM7 and the named genus
Prevotella within the Prevotellaceae (Figure 3E and Figure S2G).
Likewise, Prevotellaceae was absent from single-housed
CCL5?/?mice and highly acquired following cohousing with
NLRP6?/?mice (Figures S5J and S5K). Also included in this list
was a member of the family Helicobacteraceae (order Campylo-
bacterales); tests for the pathogen Helicobacter hepaticus were
consistently negative in these mice (n = 6 samples per strain
screened with PCR).
Histopathologic analyses of colonic sections stained with
hematoxylin and eosin as well as Warthin-Starry stain disclosed
microbes with a long branching, striated morphotype that is
closely associated with the crypt epithelium of single-housed
ASC?/?and NLRP6?/?mice; these organisms were rare in WT
mice (Figure S6F and data not shown). This morphotype is
consistent with members of TM7 (Hugenholtz et al., 2001).
Quadruple antibiotic treatment for 3 weeks eliminated microbes
with this morphology from ASC?/?mice as judged by histopath-
ologic analysis (n = 5 mice; data not shown).
A significant reduction in Prevotellaceae was noted in stools of
NLRP6?/?mice that were treated with the same combination of
four antibiotics. The most complete eradication was achieved
using a combination of metronidazole and ciprofloxacin, a
commonly used regimen for treatment of human IBD (Figure 7A).
The severity of DSS colitis was also significantly reduced in anti-
biotic-treated compared to untreated NLRP6?/?mice (Figures
7B and 7C).
Next, we tested whether antibiotic treatment affected the
ability of NLRP6?/?mice to transfer the colitogenic microbiota
to WT mice. Strikingly, WT mice cohoused with antibiotic-
treated NLRP6?/?mice developed significantly less-severe
DSS colitis compared to WT mice cohoused with untreated
NLRP6?/?mice (Figures 7D and 7E). This reduction in severity
correlated with decreased abundance of Prevotellaceae and
TM7, but not of Bacteroidetes in WT mice cohoused with antibi-
otic-treated NLRP6?/?mice (Figure 7F and Figures S6G and
S6H). Low-level representation of Prevotellaceae was noted in
nonphenotypic NLR-deficient mice bred for generations in our
vivarium (Table S1). As representative NLRs, we decided to
directly compare the quantitative differences in Prevotellaceae
abundance and its impact on transmissibility to WT mice
between NLRP6?/?and NLRC4?/?mice, as the latter lacks
a closely related colonic-epithelium-expressed protein that is
also able to form an inflammasome and process IL-18. Indeed,
NLRC4?/?and their cohoused WT cagemates featured a clus-
tering pattern in the PCoA plot (Figure 7G) distinct from both
single-housed WT mice as well as from NLRP6?/?mice and co-
housed WT mice. Specifically, Prevotellaceae was highly abun-
dant in NLRP6?/?mice though low to absent in NLRC4?/?
mice, their cohoused WT cagemates, and single-housed WT
mice (Figure 7H).
To determine whether NLRP6 deficiency was associated with
an alteration in the physical distribution (biogeography) of the
microbiota within the gut, we analyzed colon tissue that had
been thoroughly washed of fecal matter (see Experimental
Procedures for details). This enabled enhanced detection of
bacteria residing in crypts. TM7 and Prevotellaceae were
significantly more prevalent in the washed colons of NLRP6?/?
mice compared to WT and NLRC4?/?mice (Figure 7I and data
not shown). Further, transmission electron microscopy studies
revealed multiple monomorphic bacteria in crypt bases of
ASC?/?and NLRP6?/?, but not WT and NLRPC4?/?mice,
featuring an abundance of electron dense intracellular material
that is consistent with the pigmentation that is characteristic of
many Prevotella species (Figures 7J–7L and data not shown).
Overall, these findings indicate that the dysbiosis in NLRP6
inflammasome-deficient mice may involve aberrant host-micro-
bial cross-talk within the colonic crypt.
We describe a regulatory sensing system in the colon that is
dependent on the NLRP6 inflammasome. We show that genetic
deletion of components of this sensing system has drastic
leading to a shift toward a proinflammatory configuration that
drives spontaneous and induced colitis.
On a molecular level, it appears unlikely that the evolutionarily
conserved, innate mucosal immune arm possesses the ability
to distinctly identify the myriad bacterial, archaeal, and eukary-
otic microbial phylotypes and virotypes that comprise the gut
microbiota and differentiate autochthonous (entrenched) or
allochthonous (transient/nomadic) components of this commu-
nity that act as commensals or mutualists from those that act
as pathogens. Rather, this function may be achieved by sensing
signals that are related to tissue integrity or factors released by
tissue damage that serve as ‘‘danger signals’’ promoting activa-
capable of fulfilling this task, as they can be activated by many
microbial ligands, but also by host-derived factors released
upon cell or tissue damage, such as uric acid, ATP, and hyalur-
onan (Schroder and Tschopp, 2010). NLRP6 assembly in the
colonic epithelial compartment may be driven by a low level of
these substances or by yet unidentified molecules signaling
tissue integrity, resulting in local production of IL-18. Interest-
ingly, in the rat, NLRP6, caspase-1, ASC, and pro-IL-18 are
absent at embryonic day 16 (E16) and first appear at E20, with
the processed form of IL-18 emerging in the gut during the early
Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc. 753
Mass change (%)
6 10 128420
Mass change (%)
NLRP6-/- + C&M
16S rDNA Prevotellacea
16S rDNA Prevotellacea
16S rDNA Prevotellacea
(% total OTUs):
Figure 7. Decreased Abundance of Prevotella in Antibiotic-Treated NLRP6?/?Correlates with Ameliorated Colitogenic Microbiota
(A–C) WT and NLRP6?/?mice were treated with a combination of metronidazole and ciprofloxacin for 3 weeks. Prevotellaceae loads compared to total bacteria
(A) were measured in fecal samples at the end of the antibiotic treatment period using qPCR analysis. DSS exposure was begun 3 days later. Weight loss (B) and
colonoscopy score at day 7 (C).
(D–F) NLRP6?/?mice were treated with a combination of ampicillin, neomycin, vancomycin, and metronidazole for 3 weeks and then cohoused with WT mice for
4 weeks. In parallel, WT mice were cohoused with untreated NLRP6?/?mice. Subsequently, DSS colitis was induced, and weight (D) and colonoscopic
(G and H) WT mice were cohoused for 4 weeks with either NLRP6?/?or NLRC4?/?mice. (G) Unweighted UniFrac PCoA of fecal microbiota harvested after
cohousing. (H) Unweighted UniFrac PCoA colored by relative abundance of Prevotellaceae as percent of total OTUs.
(I) Quantification of Prevotellaceae in the crypt compartment, following extensive removal of stool content.
(J–L) Representative transmission electron microscopy images taken from colonic sections of WT (J, 34200), NLRC4?/?(J, 34200), NLRP6?/?(J, 32500), and
ASC?/?mice (J, 31700; K, 34200; L, 26,000).
See Figure S6 for additional evidence linking bacterial components of the gut microbiota to the transmissible colonic inflammation in NLRP6 inflammasome-
754 Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc.
postnatal period (Kempster et al., 2011), coinciding with the time
of colonization of the gut ecosystem.
Dysbiosis may contribute to IBD by expansion of colitogenic
strains such as entero-invasive E.coli (Darfeuille-Michaud
et al., 2004), by reduction of tolerogenic strains such as
Faecalibacterium prausnitzi (Sokol et al., 2008), or through a
combination of both mechanisms. In our study, a colitogenic mi-
crobiota with altered representation of distinct bacterial
members formed in the intestines of NLRP6-deficient mice;
this microbiota was transferred across generations within a
kinship and could displace the gut microbiota of cohoused
immunocompetent mice. Once this community was horizontally
transmitted to suckling or adult WT mice, it could persist.
Compared to WT mice, NLRP6 inflammasome-deficient mice
exhibit both quantitative and qualitative changes in numerous
taxa, including increased representation of members of
Prevotellaceae and TM7, and reductions in members of genus
Lactobacillus in the Firmicutes phylum.
There are several intriguing links between the abundance of
Prevotellaceae and TM7 and human diseases. Prevotellaceae
has been implicated in periodontal disease (Kumar et al.,
2003), and several reports have documented prominent repre-
sentation of this group in samples from IBD patients (Kleessen
et al., 2002; Lucke et al., 2006). Prevetellaceae might disrupt
the mucosal barrier function through production of sulfatases
that actively degrade mucus oligosaccharides (Wright et al.,
2000); these enzymes are elevated in intestinal biopsies from
IBD patients (Tsai et al., 1995). Though they have not been
cultured, members of the TM7 phylum have been identified in
16S rRNA surveys of terrestrial and aquatic microbial communi-
ties as well in human periodontal disease (Brinig et al., 2003;
Marcy et al., 2007; Ouverney et al., 2003) and in IBD patients
(Kuehbacher et al., 2008). Defining the nature of the interactions
of Prevotellaceae and TM7 with the NLRP6 inflammasome may
provide insights about probiotic interventions that may mitigate
microbiota-mediated enhanced inflammatory responses.
Four previous reports indicated that caspase-1, ASC, or
NLRP3 deficiencies were associated with an increased severity
of acute DSS colitis in mice and suggested that exacerbated
disease was mediated, in part, by a defect in repair of the intes-
tinal mucosa (Allen et al., 2010; Dupaul-Chicoine et al., 2010;
Hirota et al., 2010; Zaki et al., 2010). Opposing results were
found in two other studies using the same colitis model. The first
mation, even prior to the discovery of the inflammasome, found
ameliorated acute and chronic colitis in caspase-1?/?mice
(Siegmund et al., 2001). More recently, a second study demon-
strated reduced severity of disease in NLRP3?/?mice that
correlated with decreased levels of proinflammatory IL-1b
(Bauer et al., 2010). It has been hypothesized that these differ-
ences might be the result of distinct roles of inflammasomes
in nonhematopoietic versus hematopoietic cells (Siegmund,
2010). The proposed function in epithelial cells is to regulate
secretion of IL-18 that stimulates epithelial cell barrier function
and regeneration, whereas in hematopoietic cells, inflamma-
some activation would have a proinflammatory effect. Varying
degrees of tissue injury and subsequent inflammation may result
in shifting the balance between protective and detrimental
effects, dependingonthe experimental condition andtheinflam-
matory context. However, we believe that inflammasome-driven
effects on the colonic microbiota, as revealed in our study, add
yet another layer of regulation that affects and effects initiation
of autoinflammation. As such, exacerbation in colitis severity
in single-housed inflammasome-deficient mice may, in fact,
involve defects in tissue regeneration, but this histopathological
process may be dramatically influenced by the effects imposed
byaltered elementsin themicrobiota, including, forexample, the
enhanced representation of Prevotellaceae in the crypt. Thus,
we propose that the fundamental role of the microbiota in
shaping processes related to tissue damage, regeneration, and
stress response might offer an explanation for the opposing
results between these studies. 16S rRNA enumeration studies
combined with various permutated cohousing experiments of
the type described in this report, coupled with mechanistic
molecular studies, would allow this notion to be tested directly.
Furthermore, our results suggest that prolonged cohousing (or
littermate controls) should be used when NLRs and other innate
receptors are studied: this would allow for equilibration of differ-
ences in gut microbial ecology that may exist between groups of
mice and allow investigators to determine which features of their
phenotypes can be ascribed to the microbiota. Indeed, using
cohousing conditions, we were able to demonstrate that the
NLRC4 inflammasome is a direct negative regulator of colonic
epithelial cell tumorigenesis that is not driven by the microbiota
(Hu et al., 2010).
Our results show that the resultant aberrant microbiota
promotes local epithelial induction of CCL5 transcription as
a downstream mechanism, ultimately leading to an exaggerated
autoinflammatory response. CCL5 is potently induced by bacte-
rial and viral infections and, in turn, induces massive recruitment
of a variety of innate and adaptive immune cells carrying CCR1,
CCR3, CCR4, and CCR5 (Mantovani et al., 2004). Interestingly,
both NOD2 and TLRs have been shown to induce CCL5 tran-
scription (Be ´rube ´ et al., 2009; Werts et al., 2007). It will be of
interest to investigate the crosstalk between these immune
recognition systems in future experiments.
Recent studies have highlighted the importance of the gut
microbiota in the pathogenesis of various autoimmune disorders
that manifest outside of the gastrointestinal tract. In some auto-
immune models, germ-free conditions or inoculation with a
microbiota from healthy mice ameliorates disease (Lee et al.,
2010; Mazmanian et al., 2008; Sinkorova ´ et al., 2008; Wu et al.,
2010). In contrast, rats with collagen-induced arthritis feature
exacerbated disease when reared under germ-free conditions
(Breban et al., 1993), whereas germ-free NOD MyD88?/?mice
fail to develop diabetes, unlike their colonized counterparts
(Wen et al., 2008). In humans, epidemiological evidence points
to possible links between dysbiosis and rheumatoid arthritis,
asthma, and atopic dermatitis (Bjo ¨rkste ´n, 1999; Penders et al.,
2007; Vaahtovuo et al., 2008). Our study indicates that defi-
ciencies in the NLRP6 pathway should be added to the list of
host genetic factors that may drive disease-specific alterations
in the microbiota, which in turn may promote disease in these
communities and who have also experienced disruption in their
gut epithelial barrier function due to a variety of insults.
Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc. 755
A detailed description of materials and methods used in this paper can be
found in the Supplemental Information.
NLRP6?/?mice were generated by replacing exons 1 and 2 with a neomycin
selection cassette (IRESnlslacZ/MC1neo). For cohousing experiments, age-
and gender-matched WT and knockout mice were cohoused at 1:1 ratios for
Mice were treated with 2% (w/v) DSS (M.W. = 36,000–50,000 Da; MP Biomed-
icals) in their drinking water for 7 days followed by regular access to water.
16S rRNA Analyses
Aliquots of frozen fecal samples (n = 211) were processed for DNA isolation
using a previously validated protocol (Turnbaugh et al., 2009). ?365 bp ampli-
cons, spanningvariable region2(V2)ofthe16SrRNAgene, weregeneratedby
using primer containing barcodes and sequenced on 454 sequencer. Data
were processed using the QIIME (Quantitative Insights Into Microbial Ecology)
analysis pipeline (Caporaso et al., 2010) and analyzed using UniFrac that
defines the similarities and differences between microbial communities based
tree of life (Lozupone and Knight, 2005).
Data are expressed as mean ± SEM. Differences were analyzed by Student’s
t test and ANOVA and post-hoc analysis for multiple group comparison.
p values % 0.05 were considered significant.
16S rRNA data sets have been deposited in MG-RAST under accession
Supplemental Information includes Extended Experimental Procedures, six
figures, two data files, and one table and can be found with this article online
We would like to thank A Hafemann, S. Campton, E. Eynon, J. Alderman, W.
Philbrick, C. Zorca, M. Musaheb, J. Stein, A. Ferrandino, F. Manzo, and the
members of the Flavell lab for technical help and helpful discussions;K. Fitz-
gerald for providing AIM2?/?deficient mice; M. Graham and C. Rahner,
CCMI EM Core Facility, Yale School of Medicine; J. Manchester and S.
Deng for their assistance with DNA sequencing; V. Nagy for discussion and
help with figure preparation; and H. Elinav for important discussions, sugges-
tions, and critique. E.E. is supported by Cancer Research Institute (2010-2012)
and the Israel-US educational foundation (2009) and is the recipient of the
Claire and Emmanuel G. Rosenblatt award from the American Physicians for
Medicine in Israel Foundation. A.L.K. is the recipient of a postdoctoral fellow-
ship from the W.M Keck Foundation. J.H.-M. is supported by a LLS Postdoc-
toralFellowship. Thisworkwassupported,inpart,by HowardHughesMedical
Institute (R.A.F.) and the Crohn’s and Colitis Foundation of America (J.I.G.).
Received: December 22, 2010
Revised: March 20, 2011
Accepted: April 22, 2011
Published online: May 12, 2011
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Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc. 757
EXTENDED EXPERIMENTAL PROCEDURES
NLRP6?/?mice were generated by replacing exons 1 and 2 with a neomycin selection cassette (IRESnlslacZ/MC1neo). ASC?/?
(Pycardtm1Flv(Sutterwala et al., 2006), Casp1?/?mice (Casp1tm1Flv(Kuida et al., 1995), NLRC4?/?(Nlrc4tm1Gln, (Lara-Tejero et al.,
2006), all generated in our lab, AIM2?/?(Aim2Gt(CSG445)Byg, (Rathinam et al., 2010), kindly provided by Dr. K Fitzgerald, University
of Massachusetts), NLRP12?/?(Nlrp12tm1Jpyt, (Arthur et al., 2010), obtained from Millenium Pharmaceuticals), IL-18?/?(Il18tm1Aki,
(Takeda et al., 1998), IL-1R?/?(Il1r1tm1Imx, (Glaccum et al., 1997), both obtained from Jackson Laboratories, and IL-1b?/?mice
(Il1btm1Lvp, kindly provided by Dr. G. Sebire, CHU de Sherbrooke, (Zheng et al., 1995) were described in previous publications.
The generation of NLRP10?/?mice has not been published (S.C.E., unpublished data). NLRP6?/?, ASC?/?, Casp1?/?, and IL-
18?/?mice were backcrossed at least 10 times to C56Bl/6. IL-1R?/?mice were backcrossed five times to C56Bl/6, while IL-
1b?/?mice were on a 129S7 background (and hence used for cohousing purposes only). WT C56Bl/6 mice were purchased from
NCI. Where indicated, WT mice were also used that had been bred in our mouse barrier facility. All mice were specific pathogen-
free, maintained under a strict 12h light cycle (lights on at 7:00am and off at 7:00pm), and given a regular chow diet (Harlan, diet
#2018) ad libitum.
For cohousing experiments, age- and gender-matched WT and knockout mice were co-housed in new cages at 1:1 ratios for
4 weeks. For cross-fostering experiments, newborn mice were exchanged between ASC?/?and WT mothers within 24 hr of birth.
Mice were weaned between postnatal days 21-28. For bone marrow chimera experiments, mice were given a sublethal dose of total
body irradiation (2x 5.5 Gy, 3 hr apart). 16 hr later mice were transplanted with 4x106unseparated bone marrow cells. Mice were
analyzed 7-8 weeks later.
For antibiotic treatment, mice were given either (i) a combination of vancomycin (1 g/l), ampicilin (1 g/l), kanamycin (1 g/l), and
metronidazole (1 g/l) or (ii) a combination of ciprofloxacin (0.2 g/l) and metronidazole (1 g/l) for 3 weeks in their drinking water. All anti-
biotics were obtained from Sigma Aldrich. All experimental procedures were approved by the local IACUC.
Mice were treated with 2% (w/v) DSS (M.W. = 36,000-50,000 Da; MP Biomedicals) in their drinking water for 7 days followed by
regular access to water.
Colonoscopy was performed using a high resolution mouse video endoscopic system (‘Coloview’, Carl Storz, Tuttlingen, Germany).
The severity of colitis was blindly scored using MEICS (Murine Endoscopic Index of Colitis Severity) which is based on five param-
et al., 2006).
Colons were fixed in Bouin’s medium and embedded in paraffin. Blocks were serially sectioned along the cephalocaudal axis of the
gut to the level of the lumen; the next 5 mm-thick section was stained with hematoxylin and eosin. Each section was scored by
a pathologist who was blinded with respect to the origin of the sample: scoring was based on the degree of inflammation (location
and extent), edema, mucosal ulceration, hyperplasia, crypt loss or abscess (Hu et al., 2010; O’Connor et al., 2009). Severity scores
ranged from 0 to 5 with 0 being normal and 5 being most severe. Individual scores were assigned for each parameter, and then aver-
aged for a final score per sample. Digital light microscopic images were recorded with a Zeiss Axio Imager.A1 microscope (Thorn-
wood, NY), AxioCam MRc5 camera and AxioVision 4.7.1 imaging software (Carl Zeiss Microimaging). Results are displayed as
percent involvement of colon (inflamed colon area) and by score of the most severe lesion in each sample (pathological severity
Frozen sections of colons from WT and NLRP6?/?mice were blocked in 10% fetal bovine serum for 1 hr at room temperature. Slides
were incubated at 4?C for 16 hr with primary antibody to NLRP6 (clone E20, goat IgG, Santa Cruz Biotechnologies, Santa Cruz, Cal-
ifornia) at 2mg/ml, followed by incubation with 1:800 Alexa Fluor 647-labeled rabbit anti-goat secondary antibody (Invitrogen, Molec-
ular Probes, Eugene Oregon) for 2 hr at 4?C. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) for nuclear
staining. Slides were dried and mounted, using ProLong Antifade mounting medium (Invitrogen, Molecular Probes, Eugene Oregon).
Slides were visualized using a Leica TCS SP5 confocal microscope.
Immunoprecipitation and Western Blot Analysis
Colons were excised and washed thoroughly by flushing several times with PBS, opened longitudinally, transferred into HBSS +
2 mM EDTA, and shaken for 20 min at 37?C. Subsequently, colons were washed 3 times with PBS and these washes were pooled
with the HBSS fraction. This cell preparation containing a highly purified colonic epithelial cell fraction was spun down and resus-
pended in 1 ml / colon of ice-cold RIPA buffer containing protease inhibitors (Complete Mini EDTA-free, Roche). Cells were lysed
Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc. S1
lysates were immunoprecipated with 1 mg anti-NLRP6 antibody (clone E20, Santa Cruz Biotechnologies) and 25 ml of Protein G
agarose (Invitrogen) for 12 hr at 4?C. Agarose beads were washed five times with RIPA buffer and finally bound proteins eluted by
boiling in loading buffer. Samples were separated on 10% TGX gels (Biorad) and transferred onto PVDF membranes. Western
blot analysis was performed using a anti-NLRP6 polyclonal antibody (clone E20, Santa Cruz Biotechnologies) and anti-Goat-HRP
Isolation of Colonic CD45+Cells and FACS Analysis and Sorting
Colons were excised and washed thoroughly by flushing several times with PBS. They were opened longitudinally, transferred into
digested for 45 min at 37?C using ‘‘digest solution’’ (DMEM containing 2% FCS, 2.5mg/ml Collagenase, 1 mg/ml DNaseI, and 1mM
DTT). Single cell suspensions were obtained by grinding through a 100 mm cell strainer (Fisher Scientific). For FACS analysis, single
suspensions were stained with anti-CD11c, anti-CD11b, anti-MHC class II, anti-TCR-beta, anti-TCR-gamma/delta, anti-B220, anti-
NK1.1, and anti-CD45.2 (all from BD Biosciences or Biolegend) and analyzed on a BD LSR II. For FACS sorting, cells were stained
with anti-mouseCD45.2-PacificBlue(Biolegend) andsortedtwiceiterativelyonaBDFACSAriatoincreasethepurityofthepositively
Gene Expression Analysis
Tissues were preserved in RNAlater solution (Ambion) and subsequently homogenized in Trizol reagent (Invitrogen). Cells subjected
to FACS were resuspended in Trizol reagent. RNA was purified according to the manufacturer’s instructions. One microgram of total
RNA was used to generate cDNA (HighCapacity cDNA Reverse Transcription kit; Applied Biosystems). RealTime-PCR was per-
formed using gene-specific primer/probe sets (Applied Biosystems) and Kapa Probe Fast qPCR kit (Kapa Biosystems) on a 7500
Fast Real Time PCR instrument (Applied Biosystems). PCR conditions were 95?C for 20 s, followed by 40 cycles of 95?C for 3 s
and 60?C for 30 s. Data were analyzed using the Sequence Detection Software according the deltaCt method with hprt1 serving
as the reference housekeeping gene.
Two 0.5 cm long pieces from the proximal colon were removed from a given animal, rinsed with PBS, and weighed. The tissue
explants were cultured for 24 hr in DMEM medium containing 10% FBS, L-glutamine, penicillin, and streptomycin at 37?C. Culture
medium was removed, centrifuged (1200 x g for 7 min at 4?C), and the resulting supernatant stored in aliquots at ?20?C.
ELISA and Multiplex Analysis
Concentrations of cytokines and immunoglobulins in the serum or culture supernatants were measured using the following commer-
cial ELISA kits: CCL5 (Peprotech); IL-18 (MBL); IgG1, IgG2c (BD Biosciences), IgA, IgM (Bethyl Laboratories) according to manufac-
turer’s instruction. Multiplex analysis was performed using the Bioplex 23-Plex Panel (Biorad) according to the manufacturer’s
16S rRNA Analyses
Aliquots of frozen fecal samples (n = 212) were processed for DNA isolation using a previously validated protocol (Turnbaugh et al.,
2009). An aliquot of the purified fecal DNA was used for PCR amplification and sequencing of bacterial 16S rRNA genes. ?365bp
amplicons, spanning variable region 2 (V2) of the 16S rRNA gene were generated by using (i) modified primer 8F (50- CCATCTCA
TCCCTGCGTGTCTCCGACTCAGTCAGAGTTTGATCCTGGCTCAG-30) which consists of 454 Titanium primer B (underlined) and
the universal bacterial primer 8F (italics) and (ii) modified primer 338R (50CCTATCCCCTGTGTGCCTTGGCAGTCTCAGNNNNNNNN
code (N’s), and the bacterial primer 338R (italics). Three replicate polymerase chain reactions were performed for each fecal DNA
sample. The reactions were subsequently pooled, DNA was quantified (Picogreen), pooled in an equimolar ratio, purified (Ampure
magnetic purification beads) and used for multiplex 454 pyrosequencing (Titanium chemistry). Reads were initially processed using
the QIIME(QuantitativeInsightsIntoMicrobial Ecology) analysispipeline (Caporaso etal.,2010):fasta,qualityfilesandamapping file
indicating the barcode sequence corresponding to each sample were used as inputs. The QIIME pipeline takes this input information
taxonomic tree of the sequences based on sequence similarity, and creates a sample x OTUs table that can be used, together with
the tree,for calculating betadiversity. After chimera removal,the datasetconsistedof 747,125sequences (average numberof reads/
fecal sample, 3,524 ± 1023 (SD); average read length, 361 nt). Sequences sharing R 97% nucleotide sequence identity in the V2
region were binned into operation taxonomic units (97% ID OTUs) using uclust, chimeric sequences were removed using Chimer-
aSlayer (Haas et al., 2011). Note that we only considered 97% ID OTUs found 10 or more times among the 212 samples in our anal-
yses. The OTU table was rarefied to 100 reads per sample to normalize the depth of sequencing per sample. (The OTU table used for
our analyses is accessible at Data S1. A key describing the genotype and housing of each mouse (Table S1) shown in Figure 3 can be
found at Data S2).
S2 Cell 145, 745–757, May 27, 2011 ª2011 Elsevier Inc.