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j.celrep.2017.09.097

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Article
Distinct Microbial Communities Trigger Colitis
Development upon Intestinal Barrier Damage via
Innate or Adaptive Immune Cells
Graphical Abstract
Authors
Urmi Roy, Eric J.C. Gálvez,
Aida Iljazovic, ..., Samuel Huber,
Richard A. Flavell, Till Strowig
Correspondence
till.strowig@helmholtz-hzi.de
In Brief
Alterations in the microbiota contribute to
the development of intestinal
inflammation. Roy et al. demonstrate that
distinct intestinal microbial communities
cause colitis via opposing effector
mechanisms independent or dependent
on adaptive immunity. Their findings
suggest that personalized
immunomodulatory treatment according
to distinct microbial signatures may be
beneficial for IBD patients.
Highlights
d
Gut microbiota composition modulates colitis severity in
immunocompetent hosts
d
Colitogenic microbiota drive colitis via innate or adaptive
immunity
d
Distinct microbiota members induce pathogenic CD4+ T cells
to drive colitis
Roy et al., 2017, Cell Reports 21, 994–1008
October 24, 2017 ª 2017 The Authors.
https://doi.org/10.1016/j.celrep.2017.09.097
Cell Reports
Article
Distinct Microbial Communities Trigger Colitis
Development upon Intestinal Barrier Damage
via Innate or Adaptive Immune Cells
1 Marina C. Pils,2 Ulrike Heise,2
_
Urmi Roy,1 Eric J.C. Gálvez,1 Aida Iljazovic,1 Till Robin Lesker,1 Adrian J. B1azejewski,
Samuel Huber,3 Richard A. Flavell,4,5 and Till Strowig1,6,*
1Microbial
Immune Regulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
Pathology Platform, Helmholtz Centre for Infection Research, Braunschweig, Germany
3I. Medizinische Klinik und Poliklinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
4Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA
5Howard Hughes Medical Institute, Yale University, New Haven, CT, USA
6Lead Contact
*Correspondence: till.strowig@helmholtz-hzi.de
https://doi.org/10.1016/j.celrep.2017.09.097
2Mouse
SUMMARY
Inflammatory bowel disease comprises a group of
heterogeneous diseases characterized by chronic
and relapsing mucosal inflammation. Alterations
in microbiota composition have been proposed
to contribute to disease development, but no uniform signatures have yet been identified. Here, we
compare the ability of a diverse set of microbial communities to exacerbate intestinal inflammation after
chemical damage to the intestinal barrier. Strikingly,
genetically identical wild-type mice differing only in
their microbiota composition varied strongly in their
colitis susceptibility. Transfer of distinct colitogenic
communities in gene-deficient mice revealed that
they triggered disease via opposing pathways either
independent or dependent on adaptive immunity,
specifically requiring antigen-specific CD4+ T cells.
Our data provide evidence for the concept that microbial communities may alter disease susceptibility
via different immune pathways despite eventually
resulting in similar host pathology. This suggests
a potential benefit for personalizing IBD therapies
according to patient-specific microbiota signatures.
INTRODUCTION
Inflammatory bowel disease (IBD) consists of a complex group of
incurable inflammatory disorders comprising Crohn’s disease
(CD) and ulcerative colitis (UC). Although the etiopathogenesis
of IBD development is not fully understood, numerous studies
support the hypothesis of IBD as a pathological immune
response against microbial and environmental antigens in genetically predisposed individuals (Imhann et al., 2016; Jostins et al.,
2012). The relative contribution of innate and adaptive immune
cells and various cytokines to the development of IBD has
been controversially debated (Neurath, 2014). Nonetheless, an
imbalanced interaction between the host immune system and
gut microbiota is thought to play a pivotal role in disease manifestation and maintenance (Cho, 2008; Gevers et al., 2014;
Honda and Littman, 2012).
Notably, various human disease conditions have been associated with imbalances in the composition of the gut microbiota,
so-called dysbiosis; however, whether these changes contribute
directly to the development of the disease or reflect an altered
physiology of the host remains debated in many instances (Kamada et al., 2013; Ley et al., 2005; Turnbaugh et al., 2008).
In various mouse models of IBD, the microbiota and, in some
cases, specific members have been shown to influence disease
outcome (Saleh and Elson, 2011). Examples of IBD mouse
models that lack disease development in the absence of any microbiota are the Il10 / model of colitis and the TNFdeltaARE
model of ileitis (Keubler et al., 2015; Schaubeck et al., 2016).
Furthermore, disease development in these models is impaired
or delayed under specific pathogen-free (SPF) conditions
compared with conventional housing conditions, which potentially contain pathogenic bacteria, demonstrating that particular
microbiota members or distinct communities only present in
conventionally housed mice modulate disease onset (Laukens
et al., 2016). Specifically, Enterobacteriaceae in Tbet / Rag2 /
mice (Garrett et al., 2010) as well as Bacteroides spp. (Bloom
et al., 2011), Helicobacter spp. (Fox et al., 2011), and Bilophila
wadsworthia (Devkota et al., 2012) in Il10 / have been shown
to enhance intestinal inflammation.
The acute dextran sulfate sodium (DSS) colitis model of human
UC is considered to be largely dependent on innate immunity
(Chassaing et al., 2014). We previously demonstrated that the
dysbiotic microbiota of Nlrp6 inflammasome-deficient mice
has the ability to directly enhance DSS colitis severity, but the
effector mechanism remained unknown (Elinav et al., 2011).
Notably, a recent study identified that specific metabolites of
this dysbiotic community actively modulate innate immune
signaling and, subsequently, the host-microbiota interface
(Levy et al., 2015). Subsequently, similar dysbiotic communities
with the ability to modulate the severity of DSS colitis have been
described in other gene-deficient mice (Couturier-Maillard et al.,
2013; Hu et al., 2015; Roberts et al., 2014). However, it remains
to be examined whether different colitogenic communities
994 Cell Reports 21, 994–1008, October 24, 2017 ª 2017 The Authors.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
trigger intestinal pathologies via shared or distinct immune pathways. This knowledge could potentially explain the variable roles
that have been suggested for various immune effectors and
pathways for IBD pathogenesis.
In the present study, we have characterized the susceptibility of
mouse lines differing only in their microbiota composition toward
DSS colitis. Besides the dysbiotic community (DysN6) from
Nlrp6 / mice, interestingly, also certain but not all SPF communities demonstrated the ability to cause severe intestinal inflammation in immunocompetent mice. Strikingly, mice displayed
different inflammatory responses depending on their intestinal
microbiota composition, either characterized by infiltration of neutrophils or the presence of proinflammatory CD4+ T cells. By utilizing gene-deficient mice and antibody-mediated depletion of T cell
subsets, we demonstrated that the DysN6 community, but not
another colitogenic community, depends on CD4+ T cells to
exacerbate DSS colitis severity. Our data identify that specific interactions between colitogenic communities and host immune
pathways drive colitis development via distinct mechanisms.
RESULTS
DSS Colitis Severity Is Strongly Influenced by Microbiota
Composition in SPF Mice
Distinct differences in microbiota composition between isogenic
mice from commercial vendors—e.g., the presence of segmented
filamentous bacteria (SFB)—have been found to influence the
outcome of disease models in mice (Ivanov et al., 2009). To investigate whether C57BL/6N mice differ in their susceptibility to intestinal inflammation after chemically induced damage to the intestinal barrier, we induced DSS colitis in SPF mouse lines obtained
from vendors or bred in-house (Figure 1A; Table S1). The severity
of disease was compared within lines of SPF mice and with previously described dysbiotic Nlrp6 / mice that were obtained from
the original vivarium and subsequently bred in our animal facility
without rederivation (Figure 1B; Figure S1A; Elinav et al., 2011).
SPF-1, SPF-5, and SPF-6 mice were characterized by mild colitis
with moderate weight loss and no mortality, but SPF-2, SPF-3, and
SPF-4 mice as well as dysbiotic Nlrp6 / mice developed a similar
severe colitis with profound loss of body mass and mortality (Figure 1B; Figure S1A). Colitis severity in each representative isogenic
mouse line from different commercial or in-house sources (SPF-1,
SPF-2, SPF-4, SPF-6, and DysN6) was also illustrated by
measuring colon shortening and supported by histological characterization of tissue damage (Figures S1C and S1D). Next we
investigated fecal microbiota composition before induction of
DSS colitis using 16S rRNA gene sequencing. Analysis of b diversity using principle coordinates analysis (PCoA) showed that mice
with mild colitis severity (SPF-1, SPF-5, and SPF-6) clustered
separately from mice featuring a high severity of colitis (SPF-2,
SPF-3, SPF-4, and DysN6). We noted a high similarity between
SPF-2, SPF-3 (both from different barriers of the same vendor),
and SPF-4 mice as well as between SPF-5 and SPF-6 mice
(both from different barriers of the same vendor), respectively,
whereas SPF-1 and DysN6 mice clustered distinctly (Figure 1C).
A more detailed analysis revealed that species richness (Chao
index) was lower in SPF-1 mice but that the complexity of the community structure (Shannon index) was not significantly different
between mouse lines (Figure S1B). Global changes in the composition of microbiota have been associated with IBD (Gevers et al.,
2014), such as a decrease in the level of resident Firmicutes and/or
Bacteroides and an overabundance of Proteobacteria (Frank
et al., 2007). We observed a significant expansion of Bacteroides
over Firmicutes in colitogenic SPF-2, SPF-3, SPF-4, and DysN6
mice compared with SPF-1, SPF-5, and SPF-6 mice (Figure 1D).
Overgrowth in Proteobacteria was highest in DysN6 mice, followed by SPF-2, SPF-3, SPF-4, and SPF-5 mice, and was mostly
absent in SPF-1 and SPF-6 mice (Figure 1D; Table S2).
To exclude the effect of genetic drift in inbred mice from
different sources, we performed cohousing experiments with
microbiota donor and germ-free recipient mice. We focused on
SPF-1 (low susceptibility, higher Firmicutes), SPF-2 (high susceptibility, higher Bacteroides), and DysN6 mice (high susceptibility,
higher Bacteroides, and higher Proteobacteria) representing the
different colitis outcomes and microbiota compositions. Transfer
of the donor microbiota into germ-free (GF) recipient (exGF) mice
was confirmed by 16S rRNA gene sequencing (Figure 1E). Upon
induction of DSS colitis, exGF mice phenocopied the respective
donor mice, supporting that the differences in colitis severity
were dependent on the microbiota (Figure 1E). Similar microbiota-driven phenotypes were confirmed for the SPF-5 and
SPF-6 communities (data not shown). These data demonstrate
that distinct types of microbial communities that are stably maintained in wild-type (WT) mice are able to alter the host’s susceptibility to DSS colitis.
Transfer of Colitogenic Microbial Communities into an
Immunocompetent Host Induces Distinct Patterns of
Host Gene Expression and Alters Colitis Susceptibility
Next we investigated whether the degree of colitis severity was
also transferable between SPF mice with variable DSS colitis
susceptibility, similar to what has been observed for Nlrp6 inflammasome-deficient mice (DysN6) (Elinav et al., 2011). Therefore, we performed cohousing experiments of mice featuring
mild colitis (SPF-1) with mice having high colitis severity
(SPF-2 and DysN6). Cohousing for 4 weeks resulted in reshaping
of the microbiota in SPF-1 mice cohoused with SPF-2 mice
(SPF-1 + SPF-2) and DysN6 mice (SPF-1 + DysN6) compared
with SPF-1 control mice, respectively (Figure 2A). Moreover, cohousing also transferred colitis susceptibility (Figure 2B; Figure S2A). Because SPF-1 + SPF-2 and SPF-1 + DysN6 mice
behaved like SPF-2 and DysN6 mice, we refer to them hereafter
as cSPF-2 and cDysN6 (cohoused SPF-2 or DysN6), respectively. A similar transfer of colitis severity was also achieved by
cohousing SPF-2 with SPF-6 mice (Figure S2C) and after fecal
transplantation (FT) from SPF-2 and DysN6 mice into SPF-1
mice (data not shown). Increased colitis severity in cSPF-2 and
cDysN6 mice was also illustrated by enhanced colon shortening
and corroborated by histological characterization of tissue damage as well as endoscopy (Figures 2C and 2D; Figure S2B).
These data demonstrate that distinct types of microbial communities are able to alter the host’s susceptibility to DSS colitis even
in already colonized immunocompetent recipients.
Induction of DSS colitis has been shown to alter the composition of the intestinal microbiota (Schwab et al., 2014). To identify
whether a shared group of commensals alters their abundance
Cell Reports 21, 994–1008, October 24, 2017 995
Experimental procedure
in-house
Nlrp6-/-
Vendor 3
0
2
4
6
Time (d)
8
10
0
2
4
6
Time (d)
8
10
D
214.L11
Microbiota
101.L19
149.L19
106.L19
105.L19
143.L19
104.L19 103.L19
173.L04
142.L19 141.L19 108.L19
102.L19
162.L04
168.L04
148.L19
161.L04
151.L04
138.L19144.L04
233.L08 156.L04
176.L19
174.L19
177.L19
134.L19
173.L19
140.L19
157.L04
131.L19
159.L04 226.L08
145.L04
158.L04
150.L04
230.L08
155.L04
130.L19
136.L19 129.L19
234.L08
139.L19
135.L19 232.L08
149.L04
154.L04
227.L08
175.L19 143.L04
148.L04
225.L08
152.L04
147.L04
160.L04
137.L19
133.L19
153.L04
224.L08
220.L08
166.L04221.L08
231.L08
222.L08
216.L08
228.L08
217.L08
223.L08
229.L08
167.L04
163.L04
165.L04
164.L04
219.L08
a
a
218.L08
a
a
−0.2
a
a
a
−0.4
SPF-1
SPF-2
SPF-3
SPF-4
SPF-5
SPF-6
Nlrp6-/(DysN6)
4
2
0
139.L26
148.L26
194.L04
198.L04
195.L04
8000
4000
200
100
0
SP
F1
SP
F2
SP
FSP 3
F4
SP
F5
SP
F6
D
ys
N
6
6
212.L11
193.L04
Firmicutes/Proteobacteria ratio
211.L11
215.L11
213.L11
110.L19
217.L11
115.L19
114.L19
116.L19
113.L19
112.L19
117.L19
Axis.2 [13.8%]
0
0
o
+
+
+
o
o
+
172.L19
171.L19
159.L19
158.L19
157.L19
147.L19
175.L04
174.L04
176.L04
146.L19
100.L19
145.L19
177.L04
70
50
SPF-1
SPF-2
SPF-3
SPF-4
SPF-5
SPF-6
Nlrp6-/(DysN6)
170.L19
153.L19
0.0
80
Firmicutes/Bacteroides ratio
SP
F1
SP
F2
SP
F3
SP
F4
SP
F5
SP
F6
D
ys
N
6
156.L19
154.L19
90
155.L19
152.L19
0.2
100
100
DSS
severity
DysN6
SPF-5 SPF-6
150.L19
151.L19
10
7
Time (day)
SPF-1
C
Drinking
water
2%
DSS
SPF-4
110
Survival (%)
in-house SPF-2 SPF-3
Microbiota
B
Vendor 2
Vendor 1
Body weight (%)
A
199.L04
197.L04
155.L26 138.L26
151.L26152.L26
196.L04
140.L26
137.L26
−0.50
−0.25
0.00
0.25
Axis.1 [23.8%]
a
0.2
a
0.0
−0.2
−0.25 0.00 0.25
Axis.1 [36.9%]
Cohousing
Donor
ex-GF
S1 vs S2
110 S1 vs Dys
** *** *** *** **
** *** *** ***
100
Survival (%)
Microbiota
SPF-1
SPF-2
DysN6
0.4
Body weight (%)
Axis.2 [33.4%]
E
90
ex-GF SPF-1
ex-GF SPF-2
ex-GF DysN6
80
70
0
2
4
6
Time (d)
8
10
100
50
0
ex-GF SPF-1
ex-GF SPF-2
ex-GF DysN6
0
2
4
6
Time (d)
****
****
8
10
Figure 1. Differences in Microbiota Composition Regulate the Severity of Acute DSS Colitis
(A) DSS colitis was induced in SPF WT (SPF-1–SPF-6) and in-house bred dysbiotic Nlrp6 / (DysN6) mice by administering 2% DSS (w/v) for 7 days. Body weight
and survival of mice were examined daily for 10 days.
(B) Body weight and survival of the mice described in (A). DSS severity is depicted as ‘‘o’’ being mild and ‘‘+’’ being severe. n = 9–21 mice/group.
(C and D) Analysis of fecal microbiota composition of the mice described in (A) before DSS colitis induction using 16S rRNA sequencing. Shown is analysis of
b-diversity (PCoA) (C) and the ratio of relative abundances between Firmicutes to Bacteroides and Firmicutes to Proteobacteria (D). n = 15–33 mice/group.
(E) Germ-free C57BL/6N mice were cohoused with donor SPF WT (SPF-1, SPF-2) and Nlrp6 / (DysN6) mice, followed by induction of DSS colitis. Shown is
analysis of b-diversity (PCoA) before and disease severity (body weight and survival) upon induction of DSS colitis. n = 7–8 mice/group.
Data are displayed as mean ± SEM from at least two independent experiments. The indicated p values represent unpaired Student’s t test: *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001. See also Figure S1.
during DSS colitis in SPF-1 mice as well as in cSPF-2 and
cDysN6 mice, we compared their fecal microbial communities
before and after induction of DSS colitis (day 5). Strikingly, b-diversity analysis (PCoA) as well as an analysis of relative abundances of different bacterial families revealed minor differences
between the two time points for each community, respectively
(Figure 2E; Figure S2D). Minor alterations included an increase
in Verrucomicrobiaceae in cSPF-2 and an increase in abundance
of some Bacteroidaceae in cDysN6 (Figure 2F; Figure S2D), but
no unified changes were observed between the cSPF-2 and
cDysN6 communities despite a similar induction of colitis at
this time point. Hence, we hypothesized that colitogenic communities already modulate host immunity before disease induc-
996 Cell Reports 21, 994–1008, October 24, 2017
tion, which, in turn, results in enhancement of colitis severity.
Thus, global gene expression in colonic tissues of mice
harboring either SPF-1, cSPF-2, or cDysN6 was compared using
RNA sequencing (RNA-seq). Interestingly, SPF-1 and cSPF-2
mice clustered together and separately from cDysN6 mice with
a distinct gene expression signature (Figure 2G; Figure S2E).
Specifically, pathway enrichment analysis showed that many upregulated genes in cDysN6 were involved in T cell and B cell
signaling as well as cytokine and chemokine signaling (Figure 2H). In contrast, despite the fact that a similar colitis severity
outcome was observed in cDysN6 mice, SPF-2 colonization of
SPF-1 mice did not result in significant alterations in the host
transcriptome (Figure 2G; Figure S2E). These data together
0.25
a
a
0.00
Cohousing
None
SPF-2
DysN6
−0.25
−0.25 0.00
0.25
Axis.1 [36.9%]
110 S1 vs S1+S2
C
S1 vs S1+Dys
**** **** **** **
**** **** **** ****
10
100
Colon length (cm)
Axis.2 [33.4%]
Microbiota
SPF-1
SPF-2
DysN6
Body weight (%)
B
A
90
SPF-1
SPF-1+SPF-2
SPF-1+DysN6
80
70
0
2
4
6
Time (d)
8
10
cDysN6
SPF-1
8
7
6
SPF-1 cDysN6 cSPF-2
cDysN6 cSPF-2 SPF-1
D
9
cSPF-2
(Histology score)
15
10
5
0
SPF-1 cDysN6 cSPF-2
F
E
d0
0.4
cDysN6
SPF-1
cSPF-2
cDysN6
0.2
0.0
Treatment
-0.2
cSPF-2
Axis.2 [23.5%]
Microbiota
None
DSS
d5
Clost ridiales
Rum inococcaceae
Clostridiales/z_ot hers
Bact eroidaceae
Pept ococcaceae
Ent erococcaceae
Verrucom icrobiae
Verrucom icrobiaceae
Verrucom icrobia
Verrucom icrobiales
Odoribact eraceae
-0.4
-0.4
-0.2
0.0
0.2
Axis.1 [34.1%]
Clost ridiales/ot her
Paraprevot ellaceae
Rikenellaceae
Eubact eriaceae
6.0 4.8 3.6 2.4 1.2 0.0 1.2 2.4 3.6 4.8 6.0
LDA SCORE (log 10)
H
G
-2 -1 0 1 2 3
B Cell Receptor Signaling Pathway
Node size (# genes) Node color (p value)
EBV LMP1 signaling
IL-9 Signaling Pathway
Toll-like receptor signaling pathway
IL-4 signaling Pathway
IL-2 Signaling Pathway
Cytokines and Inflammatory Response
Inflammatory Response Pathway
p < 10
43 genes
p < 10 7
185 genes
p = 1.0
Edge width
(% shared genes)
1%
Type II interferon signaling (IFNG)
SPF-1
cSPF-2
cDysN6
Chemokine signaling pathway
T Cell Receptor Signaling Pathway
15
10 genes
Edge color
(genes from input)
6
50%
3
100%
0
Macrophage markers
Figure 2. Alteration of Colitis Susceptibility and Distinct Host Responses by Colitogenic Microbiota
(A–C) SPF-1 WT mice were cohoused with either SPF-2 WT or DysN6 Nlrp6 / (SPF-1 + DysN6) mice, resulting in SPF-1 + SPF-2 and SPF-1 + DysN6 mice,
respectively.
(A) Analysis of b-diversity (PCoA) of donor and recipient mice before induction of DSS colitis. n = 5–16 mice/group.
(B–D) Acute DSS colitis was induced, and the weight of microbiota recipient mice was monitored for 10 days (B). Colon length was measured 5 days after induction of DSS colitis. Shown is a representative image of excised colons (C). Histological analysis of distal colon was performed 5 days after induction of DSS
colitis (D). Representative pictures of H&E-stained colon sections are shown. The scale bars represent 50 mm. n = 5–16 mice/group.
(E and F) 16S rRNA sequencing of fecal microbiota from WT SPF-1, cSPF-2, and cDysN6 on day 0 and day 5 of DSS colitis. Shown are analysis of b-diversity
(PCoA) (E) and analysis of differentially abundant microbial families in cDysN6 and cSPF-2 mice on day 0 and day 5 of DSS by LEfSe (Kruskal-Wallis test, p < 0.05,
LDA 4.0) (F). n = 8–12 mice/group.
(G and H) RNA-seq analysis from total colonic tissue of WT mice colonized with SPF-1, cSPF-2, or cDysN6. The heatmap shows quantification of RNA reads (G).
Also shown is a pathway analysis based on gene ontology (GO) terms of genes significantly upregulated (2-fold) in cDysN6 mice compared with SPF-1 (H). n = 4
mice/group.
Data are displayed as mean ± SEM from at least two independent experiments. The indicated p values represent unpaired Student’s t test (B) and nonparametric
Kruskal-Wallis test (C and D): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S2.
Cell Reports 21, 994–1008, October 24, 2017 997
suggest that alteration of the SPF-1 community by colonizing
it with colitogenic SPF-2 or DysN6 triggers a very different
response at the host transcriptional level.
DysN6, but Not SPF-2, Microbiota Depends on Adaptive
Immune Cells to Develop Colitis
Because transfer of the colitogenic SPF-2 community, unlike the
DysN6 community, did not trigger large changes in the host transcriptome in the intestine, we hypothesized that the mere presence of the SPF-2 community may be sufficient to trigger more
severe colitis upon damage to the intestinal barrier. Therefore,
we assessed disease severity in SPF-1 mice that received FT
of the SPF-2 or DysN6 community 2 or 28 days prior to disease
induction, respectively. Despite minor but detectable differences
in communities of mice receiving FT for 2 or 28 days (Figures 3A
and 3C; Figure S3A), brief colonization with the SPF-2 microbiota
was sufficient to transfer exacerbated disease severity that was
comparable with the result following extended colonization (Figure 3B). In contrast, brief colonization with the DysN6 microbiota
did not transfer heightened disease susceptibility (Figure 3D).
This inability of the DysN6 microbiota to transfer colitis severity
potentially results from incomplete microbiota transfer, a
requirement for extended immunomodulation or priming of
adaptive immune responses. Comparison of the communities
in mice receiving the DysN6 FT for 2 or 28 days by linear discriminant analysis (LDA) effect size (LEfSe) analysis revealed very
minute differences (Figure 3E), including a higher abundance of
SFB as well as Odoribacteriaceae 28 days after the transfer.
Notably, despite successful transfer of colitis severity, communities differed stronger in the case of SPF-2 FT (Figure 3F). Interestingly, similar to the DysN6 FT, SFB and Odoribacteriaceae
displayed higher abundances 28 days after SPF-2 transfer.
This suggests that these bacteria may not be involved in modulating DSS colitis severity. Next, to test whether DysN6 requires
priming of adaptive immunity, we compared the severity of
DSS colitis between Rag2 / mice harboring either the SPF-1,
cSPF-2, or cDysN6 communities. Strikingly, unlike in WT mice,
cDysN6 could not enhance colitis severity in Rag2 / mice, as
indicated by similar weight loss (Figure 3G) and colon length (Figure S3D) between Rag2 / mice with SPF-1 and cDysN6. In
contrast, cSPF-2 also induced severe colitis in Rag2 / mice,
as indicated by increased weight loss and mortality (Figure 3H;
Figure S3E). Importantly, we confirmed comparable transfer of
the donor communities into WT and Rag2 / mice (Figures
S3B and S3C). We used permutational multivariate analysis of
variance (ADONIS) (Anderson, 2001), considering the variables
‘‘genotype,’’ ‘‘microbiota,’’ and ‘‘cage’’ to evaluate their relative
contribution to variability within the groups (Figures S3B and
S3C). This analysis revealed that genotype contributed only
3% of variability, whereas microbiota contributed around 60%.
Together, these data demonstrate that extended immunomodulation and priming of adaptive immunity by DysN6, but
not SPF-2, are required to exacerbate colitis severity.
Colitis Development Is Characterized by the Presence
of Distinct Immune Signatures in DysN6 and SPF-2 Mice
Despite similar disease severity in DysN6 and SPF-2 mice upon
DSS colitis induction, our initial results corroborated the hypoth-
998 Cell Reports 21, 994–1008, October 24, 2017
esis that distinct colitogenic communities contribute to disease
development via different pathways. To further compare intestinal inflammation induced in cDysN6 compared with cSPF-2
mice, the presence of cytokines and chemokines was measured
in tissue homogenates on day 7 of DSS colitis. The levels of the
pro-inflammatory cytokines interleukin-6 (IL-6) and IL-17A
were significantly higher in the distal colon of both cDysN6
and cSPF-2 mice compared with SPF-1 mice (Figure S4A).
Compared with SPF-1 and cDysN6, colitis induced in cSPF-2
mice was distinctively characterized by higher levels of interferon
g (IFN-g), IL-22, and tumor necrosis factor alpha (TNF-a) as well
as lower levels of IL-18, mainly in the distal colon (Figure S4A).
No changes were observed in IL-2, IL-4, IL-5, IL-10, and IL-13
between the three microbiota communities (data not shown). In
line with our previous observations (Elinav et al., 2011), higher
levels of the chemokine CCL5 were detected in the proximal colon of cDysN6 mice compared with SPF-1 and cSPF-2 mice (Figure S4B). In contrast, several other chemokines, including LIX
and KC, which recruit and activate neutrophils, along with
MIP-1a and MIP-1b, were significantly increased during colitis
induced by cSPF-2 (Figure S4B). In parallel, we analyzed lamina
propria leukocytes (LPLs) from colonic tissue by flow cytometry
to identify whether distinct immune cell subsets are associated
with disease induced by SPF-1, cDysN6, and cSPF-2 communities. Indeed, 2-fold increased numbers of CD45+ cells were
observed in cDysN6 WT mice compared with SPF-1 and
cSPF-2 WT mice both before and 5 days after induction of
DSS colitis (Figure 4A). In line with the enhanced levels of neutrophil-attracting chemokines, colitis in cSPF-2 mice was associated with a specific increase in the relative abundance and total
number of neutrophils (Figures 4B–4D). However, all SPF-1-,
cSPF-2-, and cDysN6-colonized mice did not demonstrate any
significant difference in disease outcome while being treated
with antibody against Ly6G compared with the isotype control
(data not shown). This might indicate a complex interaction
among different components of the innate immune system to
enhance microbiota-mediated colitis severity. Despite similar
frequencies of immune cell subsets of the adaptive immune system (Figures S4C and S4D), significant increases in the numbers
of B220+ B cells and CD3+ T cells were observed before and after
induction of DSS colitis in cDysN6 mice (Figures 4E and 4F). Increases in the numbers of CD4+ and CD8+ T cells, but not gd
T cells, contributed to this difference (Figure 4F). During, but
not before DSS colitis, a higher frequency of CD4+ T cells in
the colon of cDysN6 and cSPF-2 mice displayed an activated
phenotype (Figure S4D). Notably, the absolute numbers of activated CD4+ T cells were only increased in cDysN6 mice, both
before and after induction of DSS colitis (Figure 4F). These
analyses show that two colitogenic communities trigger distinct
inflammatory immune pathways—i.e., enhanced neutrophil
recruitment and pathogenic adaptive immune cell responses—
during DSS colitis.
ab T Cells Trigger DysN6-Mediated, but Not SPF-2Mediated, Colitis Development
To investigate which type of pathogenic adaptive immune responses contribute to disease exacerbation after colonization
with the DysN6 community, we decided to compare the severity
B
0.0
Flora transfer
No transfer
2d transfer
28d transfer
−0.3
110 S1 vs S2 (2d)
Microbiota
SPF-1
DysN6
0.1
Body weight (%)
NMDS2
70
0
2
4
6
Time (d)
Flora transfer
No transfer
2d transfer
28d transfer
0.0
−0.1
−0.2
28d
S1 vs Dys (28d)
90
0.0
NMDS1
80
2
4
6
Time (d)
0.5
F
2d
WT (SPF-1 vs cDysN6)
90
WT SPF-1
WT cDysN6
0
2
4
6
Time (d)
8
*
***
8
28d
100
***
n.s.
**
50
0
10
WT SPF-1
WT DysN6 (2d)
WT DysN6 (28d)
0
2
4
6
Time (d)
8
10
2d
4
Tenericut es
Anaeroplasm at aceae
Anaeroplasm at ales
Mollicut es
Clost ridiaceae_SFB
YS2
YS2_Ot hers
4C0d_2
Cyanobact eria
Clost ridiaceae
Odoribact eraceae
Clostridiales/ot her
5
10
Rag2-/- (SPF-1 vs cDysN6)
110
100
90
80
70
Rag2-/- SPF-1
Rag2-/- cDysN6
0
2
4
6
Time (d)
8
10
4
H
Body weight (%)
* ****
********
100
70
2
cSPF-2
2
Odoribact eraceae
Clost ridiaceae_SFB
Clost ridiaceae
1
0
1
2
3
LDA SCORE (log 10)
110
80
0
****
****
4
6
8
10
Time (d)
Porphyrom onadaceae
Body weight (%)
Body weight (%)
*
WT SPF-1
WT DysN6 (2d)
WT DysN6 (28d)
5
G
10
100
Lachnospiraceae
Rum inococcaceae
Porphyrom onadaceae
Eubact eriaceae
3
0
WT SPF-1
WT SPF-2 (2d)
WT SPF-2 (28d)
ns ns ns ns
110 S1 vs Dys (2d)
0
−0.5
4
8
50
D
0.2
cDysN6
WT SPF-1
WT SPF-2 (2d)
WT SPF-2 (28d)
80
1.0
0.3
E
90
3
2
1
0
1
LDA SCORE (log 10)
WT (SPF-1 vs cSPF-2)
110
* * *
100
Body weight (%)
C
0.0
0.5
NMDS1
100
**** **** ***
100
−0.6
−0.5
*** **** **** ***
S1 vs S2 (28d) **
Survival (%)
NMDS2
0.3
Body weight (%)
Microbiota
SPF-1
SPF-2
Survival (%)
0.6
A
90
80
70
WT SPF-1
WT cSPF-2
0
2
4
6
Time (d)
8
10
2
3
4
Rag2-/- (SPF-1 vs cSPF-2)
110
100
90
80
70
Rag2-/- SPF-1
Rag2-/- cSPF-2
0
2
4
6
Time (d)
8
10
Figure 3. The Adaptive Immune System Is Important for DysN6-Mediated, but Not SPF-2-Mediated, Colitis
(A–F) SPF-1 mice were mock-transferred or received a fecal transplant from Nlrp6 / DysN6 or WT SPF-2 donor mice 2 days or 28 days prior to colitis induction,
respectively.
(A and C) PCoA plot of fecal microbiota composition at steady state of WT SPF-1 mice receiving SPF-2 (A) or DysN6 (C) microbiota for different time periods.
n = 3–11 mice/group.
(B and D) Body weight and survival of WT SPF-1 mice receiving SPF-2 (B) or DysN6 (D) microbiota for different time periods during DSS colitis. n = 12–15 mice/
group.
(E and F) Analysis of differentially abundant microbial families in mice with short (2 days) and prolonged (28 days) exposure to DysN6 (E) and SPF-2 (F) were
analyzed by LEfSe (Kruskal-Wallis test, p < 0.05, LDA 2.0) before induction of DSS colitis. n = 3–11 mice/group.
(G and H) SPF-1 WT and SPF-1 Rag2 / recipients were cohoused with donor SPF-2 or DysN6. Body weight was monitored upon colitis induction. n = 8–12 mice/
group.
Data are displayed as mean ± SEM from at least two independent experiments. The indicated p values represent unpaired Student’s t test: *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001. See also Figure S3.
Cell Reports 21, 994–1008, October 24, 2017 999
SPF-1
SPF-1
cDysN6
cSPF-2
4.0x105
cDysN6
5.56
d0
E
24.7
1.75
cSPF-2
22.6
F
B220+ cell numbers
2.76
6.0x105
21.7
57.0
11.0
62.5
12.3
62.3
53.0
6.12
CD44
35.0
5.0x104
5
0
36.0
3.70
38.0
3.34
6.63
53.7
4.07
d0
d5
CD4+ cell numbers
2.0x105
1.0x105
1.0x10
5
5.0x104
0
0
0
d0
d5
activated CD4+ cell numbers
54.6
0
d5
CD3+ cell numbers
4.0x105
d0
5.90
1.0x105
10
2.0x105
2.0x105
CD4
neutrophil numbers
15
d0
cDysN6
2.27
D
neutrophil
20
d5
SPF-1
19.0
CD8
10.8
6.02
Ly6G
0
CD62L
cSPF-2
in total CD45+ (%)
8.0x10
C
B
CD45+ cell numbers
5
Ly6C
A
CD8+ cell numbers
4.0x10
9.0x104
d0
d5
d5
TCRgd+ cell numbers
2.0x104
4
1.0x104
6.0x104
2.0x104
5.0x103
3.0x104
0
0
0
d0
d5
d0
d5
d0
d5
Figure 4. Colitis Driven by DysN6 and SPF-2 Is Characterized by Distinct Infiltration of Innate and Adaptive Immune Cells
(A–F) Colonic lamina propria leukocytes (cLPLs) were isolated from WT mice harboring SPF-1, cDysN6, or cSPF-2 microbiota during the steady state (day 0) and
on day 5 after DSS induction and analyzed by fluorescence-activated cell sorting (FACS).
(A) Total number of CD45+ cells in cLPLs.
(B–D) Analysis of neutrophil infiltration upon DSS induction. Representative FACS plots show frequencies of neutrophils (B). Also shown are frequencies (C) and
total numbers (D) of neutrophils on day 0 and day 5 of DSS.
(E and F) Analysis of adaptive immune cells upon DSS induction. Representative FACS plots show CD4 and CD8 frequencies gated on CD3+ cells and frequencies
of naive and activated CD4+ T cells during the steady state (E). Also shown are total numbers of the indicated immune cell subsets on day 0 and day 5 of DSS (F).
Data represent 5–17 mice/group as mean ± SEM from at least two independent experiments. The indicated p values represent nonparametric Kruskal-Wallis test:
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S4.
of DSS colitis in WT as well as B or T cell-deficient mice under
SPF-1 and cDysN6 conditions. To assure comparable microbiota composition in WT and gene-deficient mice at baseline,
all gene-deficient mouse lines were initially rederived into
SPF-1 conditions using embryo transfer. To then generate
experimental cohorts of WT and gene-deficient mice, the
DysN6 microbiota was transferred into SPF-1 recipients using
FT or cohousing, and the composition of the fecal microbiota
was recorded before induction of disease. To investigate an
involvement of T and B cells, we studied SPF-1 and cDysN6
Tcrbd / and muMT / mice, respectively. Comparison of microbiota composition and multi-variate analysis before the start
of DSS colitis revealed that mice clustered according to SPF-1
and cDysN6 communities, with genotype contributing only little
(i.e., 3%) to differences in microbiome composition (Figures
S5A and S5B). Despite a similar transfer of DysN6 into WT and
Tcrbd / mice, strikingly, no difference in the severity of DSS colitis was observed between SPF-1 and cDysN6 Tcrbd / mice,
as indicated by similar weight loss, unlike in WT mice, which
showed microbiota-modulated disease severity (Figure 5A).
An involvement of T cells in transferring exacerbated disease
severity was further corroborated by analyzing intestinal inflam-
1000 Cell Reports 21, 994–1008, October 24, 2017
mation using histology (Figure 5B) and endoscopy (Figure S5C)
as well as quantifying colon shortening (Figure S5D) of WT and
Tcrbd / mice. In contrast to WT mice, deficiency in T cells resulted in no detectable differences in these parameters between
SPF-1 and cDysN6 Tcrbd / mice. Transfer of the DysN6 community into SPF-1 muMT / mice resulted in exacerbation of
DSS colitis severity, as indicated by significantly enhanced
weight loss, colon shortening, and heightened intestinal inflammation compared with SPF-1 muMT / mice, suggesting limited
involvement of B cells in colitis exacerbation (Figures 5C and 5D;
Figure S5D). To investigate whether T cells are also required for
disease exacerbation by the colitogenic SPF-2 microbiota, we
introduced the SPF-2 community into SPF-1 WT, Tcrbd / ,
and muMT / mice. After confirming that the fecal microbiota
of mice clustered according to their microbial communities and
not by genotype (Figure S5E and S5F), we induced DSS colitis.
As expected from the results with Rag2 / mice, deficiency in
B or T cells alone did not affect the transfer of heightened disease severity by cSPF-2 (Figure S5G). gd T cells have been
implicated in colonic tissue repair (Chen et al., 2002); hence,
we next characterized DSS colitis severity in SPF-1 and cDysN6
Tcrd / mice. Notably, characterization of fecal microbiota
A
B
70
0
2
4
6
Time (d)
8
10
90
80
70
Tcrbd-/- SPF-1
Tcrbd-/- cDysN6
0
2
4
6
Time (d)
8
10
C
D
0
2
4
6
Time (d)
8
10
SPF-1
80
70
muMT-/- SPF-1
muMT-/- cDysN6
0
90
80
70
WT SPF-1
WT cDysN6
0
2
4
6
Time (d)
8
10
2
4
6
Time (d)
8
10
F
Tcrd-/- (SPF-1 vs cDysN6)
110
SPF-1
100
muMT-/-
10
90
WT (SPF-1 vs cDysN6)
110
WT
cDysN6
70
WT SPF-1
WT cDysN6
100
100
90
80
70
Tcrd-/- SPF-1
Tcrd-/- cDysN6
0
2
4
6
Time (d)
8
10
5
15
cDysN6
80
E
Body weight (%)
Body weight (%)
90
Body weight (%)
Body weight (%)
100
110
10
0
SPF-1 cDysN6 SPF-1 cDysN6
WT
Tcrbd-/-
muMT-/- (SPF-1 vs cDysN6)
WT (SPF-1 vs cDysN6)
110
Histology score
WT SPF-1
WT cDysN6
100
Histology score
80
15
5
0
SPF-1 cDysN6 SPF-1 cDysN6
WT
muMT-/WT
Tcrd-/15
Histology score
90
110
SPF-1
100
Tcrbd-/-
cDysN6
110
WT
Tcrbd-/- (SPF-1 vs cDysN6)
Body weight (%)
Body weight (%)
WT (SPF-1 vs cDysN6)
10
5
0
SPF-1 cDysN6 SPF-1 cDysN6
WT
Tcrd-/-
Figure 5. ab T Cells Are Required for DysN6-Mediated Colitis
(A–F) SPF-1 WT and SPF-1 gene-deficient mice were cohoused with a DysN6 donor. Body weight was monitored upon induction of DSS colitis (A, C, and E).
Histological analysis of the distal colon was performed 5 days after induction of DSS colitis (B, D, and F). Shown are representative pictures of H&E-stained colon
sections. Scale bars represent 50 mm (B, D, and F).
(A and B) Body weight (A) and histological analysis (B) of SPF-1 and cDysN6 WT and Tcrbd / mice. n = 6–16 mice/group.
(C and D) Body weight (C) and histological analysis (D) of SPF-1 and cDysN6 WT and muMT / mice. n = 5–18 mice/group.
(E and F) Body weight (E) and histological analysis (F) of SPF-1 and cDysN6 WT and Tcrd / mice. n = 5–26 mice/group.
Data are displayed as mean ± SEM from at least two independent experiments. The indicated p values represent unpaired Student’s t test: *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001. See also Figure S5.
demonstrated that mice clustered according to SPF-1 and
cDysN6 microbiota (Figure S5H). Similar to what we observed
in WT mice, transfer of DysN6 microbiota induced in Tcrd /
mice enhanced weight loss, colon shortening, and heightened
intestinal inflammation (Figures 5E and 5F; Figure S5I). From
these results we concluded that T cells are essential for
DysN6- but not SPF-2-induced exacerbation of disease. Specifically, our data suggest that modulation of ab T cells by members
of the DysN6 community is important. Finally, we exclude a major contribution of B cells to DysN6-mediated colitis.
Pathogenic CD4+ T Cells Are Crucial to Induce DysN6Mediated Colitis
CD4+ but also CD8+ T cells contribute to different aspects of intestinal homeostasis and inflammation (Honda and Littman,
2012). Hence, we compared DSS colitis severity in SPF-1 and
cDysN6 CD4 / and CD8 / mice. Fecal microbiota of mice
clustered again according to SPF-1 and cDysN6 but not according to genotype (Figures S6A and S6B). After DSS induction,
CD8 / but not CD4 / mice showed enhanced weight loss
and colitis severity after DysN6 transfer, comparable with WT
mice (Figures 6A and 6B; Figure S6C). Furthermore, analysis of
intestinal inflammation using histology and quantification of
colon shortening (Figures 6C and 6D) in SPF-1 and cDysN6
WT and CD4 / mice corroborated that CD4+ but not CD8+
T cells are required for DysN6-induced exacerbation of disease.
To evaluate whether CD4+ T cells contribute to enhanced colitis
severity by the colitogenic SPF-2 microbiota, we introduced the
SPF-2 community into SPF-1 WT and CD4 / mice. Analysis of
fecal microbiota of mice confirmed clustering according to microbiota and not by genotype (Figure S6D). Deficiency in CD4+
T cells did not affect the transfer of heightened disease severity
Cell Reports 21, 994–1008, October 24, 2017 1001
90
80
70
WT SPF-1
WT cDysN6
0
2
4
6
Time (d)
8
100
90
80
70
10
CD8 SPF-1
CD8-/- cDysN6
-/-
0
2
4
6
Time (d)
8
100
90
80
70
10
WT
D
C
WT (SPF-1 vs cDysN6)
110
WT SPF-1
WT cDysN6
0
2
4
6
Time (d)
100
90
80
70
10
CD4-/- SPF-1
CD4-/- cDysN6
0
2
4
6
Time (d)
8
10
15
Histology score
SPF-1
9
7
cDysN6
5
SPF-1 cDysN6 SPF-1 cDysN6
WT
CD4-/-
E
Body weight (%)
100
90
80
70
WT SPF-1
WT cDysN6
0
2
4
6
Time (d)
8
5
α-CD4 Ab (SPF-1 vs cDysN6)
α-CD8 Ab (SPF-1 vs cDysN6)
isotype (SPF-1 vs cDysN6)
110
10
0
SPF-1 cDysN6 SPF-1 cDysN6
WT
CD4-/-
10
110
Body weight (%)
Colon length (cm)
8
CD4-/- (SPF-1 vs cDysN6)
110
CD4-/-
11
Body weight (%)
Body weight (%)
100
B
CD8-/- (SPF-1 vs cDysN6)
110
Body weight (%)
WT (SPF-1 vs cDysN6)
110
Body weight (%)
Body weight (%)
A
100
90
80
70
α-CD8 Ab SPF-1
α-CD8 Ab cDysN6
0
2
4
6
Time (d)
8
110
100
90
80
70
10
α-CD4 Ab SPF-1
α-CD4 Ab cDysN6
0
2
4
6
Time (d)
8
10
SPF-1
cDysN6
cSPF-2
F
CD4+ IL-17A+
CD4+ IFN-γ+
CD4+ IL-17A+IFN-γ+
1.0x103
1.0x104
0
d0
0
d5
H
pg/ml
2.0x103
d5
d0
I
95
SPF-1
cDysN6
cSPF-2
90
85
0 3
7
11
Time (d)
15
Colonoscopy score
100
10
5
0
SPF-1 cDysN6 cSPF-2
400
4000
200
0
0
d0
d5
J
15
105
1000
0
0
d0
Colon weight/length(g/cm)
2.0x10
Body weight (%)
8000
2.0x103
4
TNF-α
600
2000
3.0x104
4.0x103
IFN-γ
IL-17A
G
d0
d5
0.08
K
d5
d0
d5
CD4+ cells in colon
3.0x106
2.0x106
0.04
1.0x106
0.00
SPF-1 cDysN6 cSPF-2
0
SPF-1 cDysN6 cSPF-2
Figure 6. Pathogenic CD4+ T Cells Are Crucial to Develop DysN6-Mediated Colitis
(A) SPF-1 WT and CD8 / mice were cohoused with DysN6 donor mice, and DSS colitis was induced.
(B–D) SPF-1 WT and CD4 / mice were cohoused with DysN6 donor mice, and DSS colitis was induced. Body weight (B) during DSS colitis as well as colon
shortening (C) and intestinal inflammation (D) on day 5 of DSS colitis were monitored. Shown are representative pictures of H&E-stained colon sections. Scale
bars represent 50 mm (D). n = 5–20 mice/group.
(E) SPF-1 and cDysN6 WT mice were injected with isotype control, anti-CD8, or anti- CD4 antibodies on day 1, day 3, and day 7 of DSS colitis, and body weight
was compared. n = 10–12 mice/group.
(F) Total numbers of CD4+ T cells producing IFN-g and/or IL-17A from isolated cLPLs from IL-17AGFP IFN-gKatushka FoxP3RFP triple reporter mice with different
microbiota. n = 6–14 mice/group.
(legend continued on next page)
1002 Cell Reports 21, 994–1008, October 24, 2017
(Figure S6E), further supporting that SPF-2 drives colitis severity
irrespective of T cells.
To investigate whether pathogenic CD4+ T cells are required
during DysN6-enhanced DSS colitis, we treated SPF-1 and
cDysN6 WT mice during DSS colitis with an isotype control antibody or depleting antibodies against CD4 or CD8, respectively.
Depletion of CD4-expressing cells, but not CD8-expressing
cells, resulted in failure of the DysN6 community to exacerbate
DSS colitis (Figure 6E), highlighting that CD4+ T cells are required
during the development of DysN6-enhanced colitis.
Consequently, we extended our immunophenotyping and
analyzed the production of proinflammatory cytokines in CD4+
T cells before and during DSS colitis. We initially focused on
IFN-g and IL-17 and, hence, isolated colonic LPLs (cLPLs)
from SPF-1, cSPF-2, and cDysN6 IL-17AGFP IFN-gKatushka
FoxP3RFP triple reporter mice allowing the in situ monitoring of
cytokine production (Gagliani et al., 2015). Transfer of the
DysN6 but not SPF-2 community resulted in enhanced numbers
of IL-17A and IFN-g single and IL-17A/IFN-g double cytokineproducing CD4+ T cells already before induction of DSS colitis
(Figure 6F). After induction of DSS colitis, enhanced numbers
of cytokine-producing CD4+ T cells were observed in mice with
both colitogenic communities (Figure 6F). In addition to monitoring cytokine production in situ, we isolated cLPLs from
SPF-1, cSPF-2, and cDysN6 mice before and after induction
of DSS colitis and stimulated them with aCD3 and aCD28 to
quantify cytokine production from T cells. Strikingly, T cells
from cDysN6 mice produced larger amounts of IL-17A and
IFN-g than T cells from SPF-1 and cSPF-2 mice (Figure 6G),
both during the steady state and colitis. Notably, TNF-a production after restimulation of T cells was highest during colitis in mice
colonized with SPF-2 (Figure 6G).
To further investigate the ability of the DysN6 to drive T cellmediated intestinal inflammation, we transferred CD45RB(high)
Foxp3-CD4+ T cells from IL-17AGFPIFN-gKatushka FoxP3RFP triple
reporter mice into SPF-1, cSPF-2, and cDysN6 Rag2 / mice.
After 2 weeks, when mice differed only mildly in their weight
loss (Figure 6H), we already observed higher intestinal inflammation, as quantified by colonoscopy in cDysN6 compared with
SPF-1 and cSPF-2 recipients (Figure 6I). cDysN6 mice displayed
an enhanced colon weight to length ratio and cellular infiltration
(Figures 6J and 6K). Specifically, IFN-g+ CD4+ T cells numbers
were significantly increased (Figure S6G). Although the numbers
of IL-17A+ and double cytokine-producing T cells were also
significantly enhanced in cDysN6-colonized mice, their total
numbers were much lower than those of IFN-g+ CD4+ T cells
(Figure S6G). Taken together, this demonstrates that DysN6
induces pathogenic CD4+ T cells producing high levels of proinflammatory cytokines. Moreover, these microbiota-induced
cells are essential to drive disease in two distinct colitis models.
Recognition of Antigens from Dominant Microbial
Members by CD4+ T Cells Drives DSS Colitis Severity in
DysN6 Mice
To investigate whether recognition of microbial antigens by
CD4+ T cells is required for exacerbation of colitis in DysN6
mice, DSS colitis was induced in OTII transgenic mice colonized
with the SPF-1, cSPF-2, or cDysN6 communities (Figure S7A).
Strikingly, cDysN6 OTII mice did not display exacerbation of
DSS colitis severity, as indicated by the lack of DysN6 transferinduced changes in body weight loss, intestinal inflammation,
and colon shortening (Figures 7A and 7B; Figure S7B). In
contrast, cSPF-2 OTII mice were characterized by similar weight
loss compared with cSPF-2 WT mice (Figures S7C and S7D).
This shows that antigen specificity of CD4+ T cells is required
for modulation of disease severity by the DysN6 but not SPF-2
community.
Although the DysN6 and SPF-2 communities both trigger severe colitis in the host, the mechanisms of pathogenesis are
completely opposing. Consequently, we wanted to understand
whether triggering of innate or adaptive immunity by the SPF-2
or DysN6 community dominate over each other when cotransferring them into SPF-1 recipients. Analysis of microbiota composition in recipient mice after cohousing of SPF-1 recipient as
well as SPF-2 and DysN6 donor mice showed that the resulting
community largely resembled the cDysN6 community (Figure 7C). Accordingly, the host gene expression signatures in
cDysN6+SPF-2 mice were similar to the ones observed in
cDysN6 mice, including upregulation of genes associated with
T cell, B cell, cytokine, and chemokine signaling as well as upregulation of Cd4 (Figure 7D; Figures S7E and S7F). Moreover,
mice with cDysN6+SPF-2 displayed high weight loss, intestinal
inflammation, colon shortening, and mortality compared with
SPF-1 mice (Figure 7E; Figures S7G and S7H). Strikingly, the
cDysN6+SPF-2 community failed to induce severe colitis in
CD4 / mice (Figure 7F; data not shown). These results demonstrate that the DysN6 community and its pathogenesis mechanism (i.e., the induction of pathogenic antigen-specific CD4+
T cell responses) dominate over SPF-2-induced changes during
colitis induction.
DISCUSSION
Alterations in the microbiome have been hypothesized to
contribute to the development of IBD, and patient data suggest
the existence of microbial signatures associated with specific
disease entities such as CD (Frank et al., 2007; Gevers et al.,
2014). However, it remains in question whether those changes
are causal and can be potentially used to improve the selection
of IBD therapy or, rather, are the result of ongoing inflammation
that alters the intestinal microenvironment (Börnigen et al.,
(G) cLPLs were isolated from SPF-1, cDysN6, and cSPF-2 mice during the steady state and on day 5 after DSS colitis induction and restimulated with a-CD3/
CD28 for 3 days. Cytokine levels were measured from supernatant. n = 5 mice/group.
(H–K) T cell transfer colitis was induced by injecting CD4+Foxp3 CD45RB(high) T cells into SPF-1, cDysN6, or cSPF-2 Rag2 / recipients. Body weight was
measured after T cell transfer (H). Shown is the colonoscopy severity score on day 14 after transfer (I). Colon weight/length ratio (J) and total numbers of CD4+
cells in cLPLs on day 16 after injection were monitored by FACS (K). n = 7–14 mice/group.
Data are displayed as mean ± SEM from at least two independent experiments. The indicated p values represent unpaired Student’s t test (A–E) and
nonparametric Kruskal-Wallis test (F–K): *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S6.
Cell Reports 21, 994–1008, October 24, 2017 1003
A
B
70
WT SPF-1
WT cDysN6
0
2
4
6
Time (d)
8
100
Histology score
90
SPF-1
100
15
110
90
80
70
10
OTII SPF-1
OTII cDysN6
0
2
4
6
Time (d)
8
10
C
cDysN6
110
80
OTII
WT
OTII (SPF-1 vs cDysN6)
Body weight (%)
Body weight (%)
WT (SPF-1 vs cDysN6)
D
Macrophage markers
Node size (# genes) Node color (p value)
SPF-1
cDysN6
-0.2
cSPF-2
Chemokine signaling pathway
cDysN6+SPF-2
-0.4
-0.6
-1.2
Inflammatory Response Pathway
-0.8
-0.4
0.0
NMDS1
0.4
F
100
Survival (%)
100
90
SPF-1
cDysN6+SPF-2
0
2
4
6
Time (d)
8
10
10 genes
p < 10
91 genes
p < 10 6
831 genes
p = 1.0
Edge width
(% shared genes)
13
Edge color
(genes from input)
1%
5
50%
2
100%
0
T Cell Receptor Signaling Pathway
WT (SPF-1 vs cDysN6+SPF-2)
110
80
EBV LMP1 signaling
B Cell Receptor Signaling Pathway
Body weight (%)
NMDS2
XPodNet - protein-protein interactions
in the podocyte expanded by STRING
-
0.0
Body weight (%)
Type II interferon signaling (IFNG)
Microbiota
0.2
70
5
0
SPF-1 cDysN6 SPF-1 cDysN6
WT
OTII
0.4
E
10
50
0
SPF-1
cDysN6+SPF-2
0
2
4
6
8
Time (d)
10
CD4-/- (SPF-1 vs cDysN6+SPF-2)
110
100
90
80
CD4-/- SPF-1
CD4-/- cDysN6+SPF-2
70
0
2
4
6
Time (d)
8
10
Figure 7. CD4+ T Cells Drive DSS Colitis Severity in DysN6 Mice by Recognizing Antigens from Dominant Microbial Members
(A and B) SPF-1 WT and OTII transgenic mice were cohoused with DysN6 donor mice, and DSS colitis was induced. Body weight (A) and intestinal inflammation
(B) on day 5 of DSS colitis were monitored. Shown are representative pictures of H&E-stained colon sections. Scale bars represent 50 mm. n = 5–10 mice/group.
(C–E) SPF-1 WT mice were cohoused with DysN6, SPF-2, or both DysN6 and SPF-2 donor mice, respectively. Shown is b-diversity analysis (PCoA) of fecal
microbiota (C) and pathway analysis based on GO terms of genes significantly upregulated (2-fold), as determined by RNA-seq in cDysN6+SPF-2 compared with
SPF-1 mice (D). Also shown are body weight loss and survival after induction of DSS colitis (E).
(F) SPF-1 CD4 / mice were cohoused with DysN6 and SPF-2 donor mice, and DSS colitis was induced. n = 5–12 mice/group.
Data are displayed as mean ± SEM from at least two independent experiments. The indicated p values represent unpaired Student’s t test: *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001. See also Figure S6.
2013). Here we identified and then characterized distinct types of
microbial communities that directly affect the severity of intestinal inflammation in an immunocompetent host. We showed
that these communities alter disease susceptibility via opposing
mechanisms, one requiring antigen-specific CD4+ T cell responses and the other being mediated by innate immune cells.
Healthy human individuals can differ greatly in the composition
of their intestinal microbiota, but despite this variability, alterations in the microbiota of patients have been associated with
different types of human diseases (Clemente et al., 2012; Falony
et al., 2016). Germ-free mice have been fundamental to address
the causal role of alterations of the microbiome in mouse models
of human disease. Likewise, conventionally housed laboratory
mice that feature tremendous differences in the microbiome
represent a valuable resource to study the contribution of diverse
microbial ecosystems to disease development (Stappenbeck
1004 Cell Reports 21, 994–1008, October 24, 2017
and Virgin, 2016). In a first effort to reduce experimental variability, the concept of SPF housing conditions was introduced
to exclude unwanted influences imposed by the presence of potential pathogens, such as Helicobacter spp. or mouse norovirus, commonly present in wild and conventionally housed mice
(Stappenbeck and Virgin, 2016). However, microbiota composition differs greatly between SPF mice from different commercial
breeders and academic institutions (Rausch et al., 2016), and
those differences influence host responses; e.g., the presence
of Th17 cells in SFB-colonized mice (Ivanov et al., 2009) or lowered susceptibility to malaria infection as a consequence of an
increased abundance of Lactobacillaceae and Bifidobacterium
spp. (Villarino et al., 2016). These observations also make genetically identical SPF mice a versatile experimental model to
explore diverse microbial communities and to study host-microbiota interactions in health and disease.
A common feature in IBD, particularly in UC, is impairment of
the intestinal barrier, resulting in enhanced exposure to luminal
microbes. By employing a mouse model of damage to the intestinal barrier, DSS colitis, we demonstrate that isogenic SPF mice
with differences in microbiome composition feature altered susceptibility to intestinal inflammation. Specifically, we noted that
transfer of colitogenic communities into mice relatively resistant
to induction of DSS colitis is sufficient to alter disease susceptibility even in immunocompetent mice. Upon induction of disease, the DysN6 community as well as the SPF-2 community
induced severe colitis compared with the relatively resistant
SPF-1 community, but the mechanisms of pathogenesis differed
strongly. Inflammation in SPF-2 mice was characterized by high
levels of TNF-a and neutrophil-attracting chemokines coinciding
with significant higher infiltration of neutrophils into the inflamed
tissue. In line with previous findings, DysN6 mice featured higher
levels of the chemokine CCL5, known to attract innate and adaptive immune cells carrying CCR1, CCR3, CCR4, and CCR5 (Elinav et al., 2011). Here we identified high infiltration of activated
CD4+ T cells in DysN6 mice, hinting toward a potential involvement of these cells in intestinal pathogenesis. The hypothesis
of adaptive immune cells being involved in DysN6 mice was
further corroborated by the observation that extended colonization with the DysN6 but not SPF-2 community was required
to transfer disease susceptibility. Subsequently, we evaluated
the effect of the transfer of the two colitogenic communities
in mice lacking specific subsets of adaptive immune cells. For
these comparisons we employed WT and gene-deficient mice
that were embryo-transferred into our vivarium using SPF-1 foster mothers, resulting in a standardized microbiota (E.J.C.G,
unpublished data). Moreover, we included cohousing of WT
and gene-deficient mice to further reduce microbiota variability
within experiments and documented, for all experiments, microbiota composition using 16S rRNA gene sequencing. Using this
carefully controlled approach, we observed significant increases
in IL-17A and IFN-g secretion by CD4+ T cells during DysN6- and
SPF-2 driven colitis. Notably, this is in line with an association of
CD4+ T cells and proinflammatory cytokines, including IL-17,
IFN-g, and IL-23, with human IBD (Kaser et al., 2010). Strikingly,
our experiments demonstrated that CD4+ T cells are only essential to mediate the exacerbation of DSS colitis in DysN6 but not
SPF-2 mice. In contrast, despite measurable CD4+ T cell activation during DSS colitis, SPF-2 modulated disease severity independent of adaptive immune cells. T cell receptor (TCR)-mediated recognition of cognate antigens is required for proper
T cell function, and recognition of microbial antigens has been
suggested to significantly contribute to the development of colitis (Feng et al., 2010). Using OTII transgenic mice, we could show
that DysN6-driven but not SPF-2-driven colitis development
strongly depended on the presence of antigen-specific CD4+
T cells. The presence of in vivo cytokine-secreting CD4+ T cells
before induction of DSS colitis in DysN6 mice suggests that
colonic CD4+ T cells already recognize cognate microbial antigens during this phase, similar to what has been observed for
SFB-specific CD4+ T cells in the small intestine (Yang et al.,
2014). Importantly, antibody-mediated depletion of CD4+
T cells during colitis resulted in failure to transfer enhanced colitis
susceptibility. This demonstrated that, to enhance colitis, modu-
lation of the mucosal barrier by CD4+ T cells in the steady state
was not sufficient and, rather, required the presence and, presumably, the effector functions of CD4+ T cells during colitis.
The distinct property of the DysN6 community to prime and activate pathogenic CD4+ T cell responses was further corroborated
using a model for CD4+ T cell-mediated colitis. Specifically,
transfer of CD4+ T cells in Rag2 / mice harboring the DysN6
but not the SPF-2 microbiota enhanced intestinal inflammation
and cytokine production by CD4+ T cells. Whether these different
communities also cause different disease susceptibility or pathogenesis via shared or distinct pathways in other inbred mouse
strains or IBD models such as the Il10 / model of colitis and the
TNFdeltaARE model of ileitis, remains to be tested (Keubler et al.,
2015; Schaubeck et al., 2016). This shows that colitogenic communities exert their pathogenic effects in the same disease
model by opposing mechanisms.
Detailed characterization of the colitogenic communities using 16S rRNA gene sequencing revealed the varying presence
of potential pathobionts such as SFB, Prevotella spp., Helicobacter spp., Enterobacteriaceae, and Verrucomicrobiaceae in
DysN6 and SPF-2 mice. SFB have been shown to modulate intestinal T cell immunity and systemic autoimmunity (Ivanov
et al., 2009). However, based on their presence in both SPF-2
and DysN6 mice, a role in driving the differential requirement
for CD4+ T cells can be excluded. Similarly, members of the
genus Prevotella, previously found to be enriched in the colitogenic microbiota of Nlrp6 / mice (Elinav et al., 2011), were present in both colitogenic communities, indicating that they are
not involved in regulating the different pathogenicity modes.
Helicobacteraceae have been demonstrated to induce the
development of colitis in Il10 / mice in cooperation with other
members of the microbiota (Keubler et al., 2015). Although
Helicobacteraceae were absent in SPF-2 mice, DysN6
mice harbored different members of this family, including
H. typhlonius, H. rodentium, and H. muridarum, but did not harbor H. hepaticus. Finally, both Enterobacteriaceae and Verrucomicrobiaceae, specifically Akkermansia muciphilia, bloomed
during induction of DSS colitis in SPF-2 mice, but it is being
debated whether expansion during disease suggests a
contribution to disease development or, rather, a consequence
of the ability to utilize inflammation-induced metabolites.
In contrast to the ‘‘one microbe one disease’’ model, the
concept of dysbiosis, an imbalance of the community, has
been proposed for microbiome-mediated modulation of
diseases (Petersen and Round, 2014). One characteristic of
dysbiotic communities, including those in IBD patients, has
been suggested to be an imbalance between Bacteroides,
Firmicutes, and Proteobacteria, with an overexpansion of
Bacteroides and Proteobacteria over Firmicutes (Frank et al.,
2007). Lowered Firmicutes/Bacteroides ratios were noted in
all colitogenic communities, including SPF-2 and DysN6,
whereas the ratios between Firmicutes and Proteobacteria
(F/P) was not consistently different between susceptible and
resistant groups. Notably, the F/P ratio was the lowest in
DysN6 mice, and according to our data, this is associated
with a distinct mode of pathogenicity. Whether, in the cases
of the SPF-2 and DysN6 community, specific pathobionts or
a general dysbiosis are responsible for driving distinct
Cell Reports 21, 994–1008, October 24, 2017 1005
pathogenicity requires further investigation because dysbiotic
communities have also been reported in other gene-deficient
mouse lines (Couturier-Maillard et al., 2013; Hu et al., 2015;
Roberts et al., 2014).
Finally, it remains debated how these dysbiotic communities
arise in gene-deficient mice and how similar they are in regard
to their composition in different vivariums, taking into account
that a large variability in the composition and function
of microbial ecosystems in WT mice already exist. In this
study, we employed Nlrp6 / mice with a similar microbiome
compared with what has been reported previously (Elinav
et al., 2011). However, it remains to be tested whether the
pathological mechanism of dysbiotic communities occurring
in unrelated lines of Nlrp6 inflammasome-deficient mice
causes exacerbated pathology via the same or different mechanisms or does not cause any pathology at all. Along these
lines, a recent study has suggested that the intestinal microbiomes of WT and Nlrp6 / mice raised under SPF conditions
did not differ in their composition, suggesting that the development of dysbiotic communities reflects a complex interplay
between genetic and environmental factors (Mamantopoulos
et al., 2017).
In summary, our data show how distinct microbial communities drive the development of intestinal inflammation in immunocompetent hosts by modulating opposing arms of the immune
system. Our study suggests the concept that triggering of
different immune pathways by microbial communities can alter
disease susceptibility, eventually resulting in similar host pathophysiology. This implies that personalized immunomodulatory
treatment according to distinct microbial signatures may be
beneficial for IBD patients.
(Caporaso et al., 2011). Samples were sequenced on an Illumina MiSeq platform (PE250). Filtering of sequences for low-quality reads (q > = 30) and barcode-based binning were performed by using QIIME v1.8.0 (Caporaso et al.,
2010). Reads were clustered into operational taxonomical units (OTUs) based
on 97% nucleotide identity of the amplicon sequences using UCLUST reference OTU picking, followed by taxonomic classification using the Ribosomal
Database Project (RDP) classifier executed at 80% bootstrap confidence cutoff (Edgar, 2010; Wang et al., 2007). Sequences without a matching reference
dataset were grouped as de novo using UCLUST. The OTU absolute abundance table and mapping file were used for statistical analyses and data visualization in the R statistical programming environment package phyloseq
(McMurdie and Holmes, 2013). To determine bacterial OTUs that explained
differences between microbiota settings, the LEfSe method was used (Segata
et al., 2011). OTUs with Kruskal-Wallis test < 0.05 and LDA scores > 3.5 were
considered informative. Raw data are available in the Sequence Read Archive
(SRA): PRJNA407363.
DSS-Induced Colitis
To induce acute colitis, mice were provided 2% (w/v) DSS (molecular
mass = 36–50 kDa, MP Biomedicals) in drinking water for 7 days, followed
by 7 days of access to regular drinking water. Daily clinical assessment of
DSS-treated animals included body weight loss measurement, stool consistency, and detection of blood in the stool. Experimental samples were
collected on days 0, 5, and 7 of DSS treatment.
CD45Rbhi Colitis
CD4+Foxp3 CD45RB(high) cells were transferred adoptively into Rag2 /
mice according to the protocol described by Ostanin et al. (2009). Briefly,
splenic lymphocytes were isolated from IL-17AGFP IFN-gKatushka FoxP3RFP
triple reporter mice. CD4 enrichment was performed according to the
manufacturer’s instructions using CD4 (L3T4) microbeads (Miltenyi Biotec).
CD4-enriched cells were then stained with antibodies against CD45RB and
CD4. Cells were sorted in a BD FACSAria II cell sorter by gating CD45RB(high),
CD4+Foxp3 cells. Antibodies used for staining were anti-CD45RB (C36316A) and anti-CD4 (GK1.5). A total of 500,000 cells were injected intraperitoneally (i.p.) per mouse. Disease development was monitored by weighing animals
3 times a week and performing colonoscopies.
EXPERIMENTAL PROCEDURES
Mice
Wild-type and all transgenic mice, Rag2 / , Tcrbd / , muMT / , Tcrd / ,
OTII, CD4 / , CD8 / , and IL-17AGFP IFN-gKatushka FoxP3RFP reporter mice
used in the study were on the C57BL/6N background, rederived into SPF-1
microbiota by embryo transfer, and bred at the SPF animal facilities of the
Helmholtz Centre for Infection Research (HZI). Nlrp6 / mice were obtained
from Yale University and subsequently bred under conventional housing conditions at the HZI without rederivation. Other donor microbiota for different
composition were purchased from different commercial vendors (Janvier,
Taconic, and Harlan) (Table S1). Germ-free C57BL/6NTac mice were bred in
isolators (Getinge) in the germ-free facility of the HZI. All experiments were
performed with 10- to 14-week-old age-matched and gender-matched
animals. Both male and female animals were used for every experiment to
exclude influence of gender.
Statistical Analyses
Statistical analysis was performed using the GraphPad Prism program
(GraphPad). Data are expressed as mean ± SEM. Differences were analyzed
by Student’s t test and ANOVA. The indicated p values represent non-parametric Mann-Whitney U test or Kruskal-Wallis test comparison between
groups. p Values % 0.05 were considered significant: *p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001.
DATA AND SOFTWARE AVAILABILITY
The accession number for the RNA-seq data reported in this paper is
Sequence Read Archive (SRA): SRP118483. The accession number for the
16S rRNA gene sequencing data reported in this paper is SRA: SRP119278.
SUPPLEMENTAL INFORMATION
DNA Isolation and 16S rRNA Microbial Community Analysis
Fresh stool samples of mice were collected and immediately stored at 20 C.
DNA was extracted according to established protocols using a method
combining mechanical disruption (bead-beating) and phenol/chloroformbased purification (Turnbaugh et al., 2009). Briefly, a sample was suspended
in a solution containing 500 mL of extraction buffer (200 mM Tris, 20 mM
EDTA, and 200 mM NaCl [pH 8.0]), 200 mL of 20% SDS, 500 mL of phenol:
chloroform:isoamyl alcohol (24:24:1), and 100 mL of 0.1 mM zirconia/silica.
Samples were homogenized twice with a bead beater (BioSpec) for 2 min.
After precipitation of DNA, crude DNA extracts were resuspended in Tris,
EDTA (TE) buffer with 100 mg/mL RNase and column-purified to remove
PCR inhibitors (BioBasic). Amplification of the V4 region (F515/R806) of the
16S rRNA gene was performed according to previously described protocols
1006 Cell Reports 21, 994–1008, October 24, 2017
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and two tables and can be found with this article online at
https://doi.org/10.1016/j.celrep.2017.09.097.
AUTHOR CONTRIBUTIONS
U.R. and T.S. designed the experiments and wrote the manuscript with input
from co-authors. U.R., A.I., and A.J.B. performed and analyzed the experiments. E.J.C.G. analyzed the RNA sequencing data. E.J.C.G. and T.R.L. designed and supported the analysis of the 16S rRNA sequencing data.
M.C.P. and U.H. performed histological evaluation and analysis. R.A.F.
contributed essential reagents. S.H., R.A.F., and T.S. supervised the study.
ACKNOWLEDGMENTS
We thank the members of the Strowig, Huber, and Flavell laboratories, as well
as Nicola Gagliani, for valuable discussions. We thank Achim Gronow, Annett
Kluge, the staff of the animal unit, and the genome analytics core facility at the
Helmholtz Centre for Infection Research for excellent technical support. The
project was supported by the Helmholtz Association (VH-NG-933 to T.S.),
by the DFG (STR-1343/1 and STR-1343/2 to T.S.), and the EU (MCCIG618925
to T.S. and StG337251 to S.H.).
Received: May 23, 2017
Revised: August 15, 2017
Accepted: September 28, 2017
Published: October 24, 2017
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