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! 1
Supplementary Materials 1! 2!Supplementary figures 3!
4!Supplementary figure 1. Modulation in cytokine production in DSS treated Il33-/- mice. 5!Quantification of indicated cytokines in the colon explants of WT and Il33-/- mice at indicated 6!days post DSS administration. Similar data was obtained with the sera (not shown). Data 7!represent two independent experiments and analyzed by Mann-Whitney U test. Error bars 8!represent mean±S.E.M with 10 mice per group per time point. 9! 10! 11!
12!
13!
14!
15!
16!
17!
18!
19!
20!
21!
22!
23!
24!
25!
Day 4 Day 80.0
0.5
1.0
1.5
2.0
IP-1
0 (n
g/m
l)
*NSWTIl33-/-
Day 4 Day 80
5
10
15
20
TNF-α
(pg/
ml)
NS NS
Day 4 Day 80
25
50
75
100
125
IL-1
7 (p
g/m
l)
NS NS
Day 4 Day 80
20
40
60
80
CC
L5 (p
g/m
l)
NS NS
Day 4 Day 80.0
0.2
0.4
0.6
0.8
1.0
GM
-CSF
(ng/
ml) NS NS
! 2
26!
Supplementary figure 2. Levels of IL-33 and IL-1α are increased in the colon after DSS 27!administration. WT and Il33-/- mice were treated with DSS in drinking water for 6 days, followed 28!by drinking water for 2 days. (A) Western blot analysis for IL-33 in the colon lysates. (B) qRT-29!PCR for Il33 expression in the colon tissue. (C) qRT-PCR analysis of Il33 and Il1α expression 30!from indicated cell populations from the epithelial (Ep) and colonic lamina propria (cLP) fractions 31!of WT mice at day 8. (D) Western blot analysis for caspase-1 in colon lysates. (E) H&E staining 32!at 40x original magnification of colon sections at day 4 post DSS. Data represent two 33!
Ep Epc
am+
Ep CD45
+
cLP C
D90+
cLP M
HCII+02468
200400
Il1α
R
elat
ive
expr
essi
on *** *** *** ***
Ep Epc
am+
Ep CD45
+
cLP C
D90+
cLP M
HCII+0
5
10
15
20
25
Il33
Rel
ativ
e ex
pres
sion
WaterDSS
N.D.
*** *** ***
0
2
4
6
8
10
Il33
Rel
ativ
e ex
pres
sion
0 4 8Days post DSS
***A B
C
WT Il33-/-
D
E
IL-33 GAPDH
Day 8 Day 0
WT Il33-/- Day 4
caspase-1 p10
caspase-1 p 45
Short Exposure
Long Exposure
Day 8 WT Il33-/-
! 3
independent experiments and analyzed by Mann-Whitney U test. Error bars represent 34!mean±S.E.M with 5 mice per group per time point. 35!
36!
37!
38!
39!
40!
! 4
41!Supplementary figure 3. IL-1α promotes colitis and associated cancer. (A) Survival and (B) 42!disease Activity Index of WT and Il1α-/- mice administered 4% DSS in drinking water. (C) Body 43!weight change of WT and Il1α-/- mice injected with AOM on day 0 and administered 5 rounds of 44!3.5% DSS in drinking water. (D) and (E) Colon histology analysis and (F) proportion of mice with 45!low- or high-grade epithelial dysplasia at day 108 post AOM injection. (G) H&E staining at 10x 46!original magnification of the distal and middle colon sections at day 108 post AOM injection. 47!
5 11 26 32 41 47 63 69 72 86 89 92
90
105
120
135
Days post AOM
Initi
al b
ody
wei
ght (
%) WT (n=10)
Il1α-/- (n=11)
*****
DSS DSS DSS DSS DSS
**
WT Il1α-/-0
5
10
15
20
Tota
l his
tolo
gica
l sco
re *
WT Il1α-/-0
50
100
150
% o
f mic
e w
ith d
yspl
asia
Low gradeHigh grade
Inflam
mation
Ulcerat
ion
Hyperp
lasia
Area01234567
His
tolo
gica
l sco
re
*
0 4 8 12 16 200
20
40
60
80
100
Days post DSS
Surv
ival
(%)
4% DSS
WT (n=9)Il1α-/- (n=12)
**
0 3 5 7 8 90
1
2
3
4
5
Days post DSS
Dis
ease
Act
ivity
Inde
x ********
0 5 8 11 12 14 15 26 29 32 33 35 41 44 47 48 49 63 66 69 70 72 83 86 88 89 90 9280859095
100105110115120125130135
WT (n=7)IL-1a-/- (n=6)
** *** *
*******
**
Days after AOM
Bod
y W
eigh
t Cha
nges
(%)
WT IL-1a-/-0
5
10
15 p=0.09
Num
ber o
f tum
or p
er c
olon
Inflam
mation
Ulcer
Hyperp
lasia
Area0
2
4
6
8 WT IL-1a-/- *
His
tolo
gica
l sco
res
WT IL-1a-/-0
10
20
30
40
*
Tota
l his
tolo
gica
l sco
res
D0 Colo
n
D14 C
olon
D80 N
on-tu
mor co
lon
D80 Tum
ors0.000
0.001
0.002
0.003
**
****
*IL-1a
mR
NA
/ beta-actin
<2mm 2-4mm >4mm0
20
40
60
80WT IL-1a-/-
% o
f tum
or s
ize
(dia
met
er)
WT IL-1a-/-0
50
100
150 Low grade dysplasiaHigh grade dysplasia
Per
cent
age(
%) o
f mic
e w
ith d
yspl
asia
A B
C D E
F G
Figure 1
HWT IL-1α-/- WT IL-1α-/-
Middle colon Rectum G WT Il1α-/-
Middle Colon WT Il1α-/-
Distal colon
A B
C
D E F
! 5
Data represent two independent experiments and analyzed by two-way ANOVA followed by 48!Holm-Sidak post test (B and C) or Mann-Whitney U test (D and E). Error bars represent 49!mean±S.E.M. 50!
51!
52!
53!
54!
55!
56!
57!
58!
59!
60!
61!
62!
63!
64!
65!
66!
67!
68!
69!
70!
71!
72!
73!
74!
75!
76!
77!
78!
79!
80!
81!
82!
! 6
83!Supplementary figure 4. Modulation of goblet cells during DSS administration in Il33-/- 84!mice. (A) Quantification of expression of mucins and goblet cell associated genes in the colons 85!of WT and Il33-/- mice at indicated days post DSS administration by qRT-PCR. (B) 86!Quantification of the number of goblet cells per crypt. (C) PAS staining at 10x original 87!magnification of colon sections. Data was analyzed by Kruskal-Wallis test followed by Dunn’s 88!post test. Error bars represent mean±S.E.M. N= 8 each for (A), N=5 each for (B) and (C). Each 89!dot represents an individual crypt. 90!
91!
92!
93!
94!
95!
96!
97!
98!
99!
100!
101!
024
100
200
Muc2
Rel
ativ
e ex
pres
sion ****NS NS
Day 0 Day 8Day 40
1
2
3
4Muc1
Rel
ativ
e ex
pres
sion NS NS NS
Day 0 Day 8Day 4
WTIl33-/-
0.0
0.5
1.0
1.5
2.0
Muc3
Rel
ativ
e ex
pres
sion **NS NS
Day 0 Day 8Day 4
012345
Muc4
Rel
ativ
e ex
pres
sion NS NSNS
Day 0 Day 8Day 40
2
4
6
8
Muc5ac
Rel
ativ
e ex
pres
sion ***NS NS
Day 0 Day 8Day 40
2
4
6
Tff3
Rel
ativ
e ex
pres
sion NS NSNS
Day 0 Day 8Day 4
0
10
20
30
Gob
let c
ells
per
cry
pt NS
Day 0 Day 8Day 4
*** ****
A
B
WT
Il33-/-
Day 0 Day 4 Day 8 C
! 7
102!Supplementary figure 5. ILC2 analysis in the colons of WT and Il33-/- mice. (A) 103!Quantification of expression of type2 cytokines in the colons of WT and Il33-/- mice at indicated 104!days post DSS administration by qRT-PCR. (B) Gating strategy for ILC2 cells, where Lin- 105!represents CD3-B220-Ly-6C-Ly-6G-CD11b-Ter119-NKp46-. Proportion and total number of (C) 106!ILC2s and (D) TH2 cells in the lamina propria. Data represent two independent experiments and 107!analyzed by Kruskal-Wallis test followed by Dunn’s post test. Error bars represent mean±S.E.M. 108!with 10 mice per group per time point. 109!
110!
111!
112!
113!
0
5
10
15
Amphiregulin
Rel
ativ
e ex
pres
sion ****NS NS
Day 0 Day 8Day 40
1
2
3
4Il5
Rel
ativ
e ex
pres
sion ***NS NS
WTIl33-/-
Day 0 Day 8Day 40
5
10
15
Il13
Rel
ativ
e ex
pres
sion ****NS NS
Day 0 Day 8Day 4
0
500
1000
1500
2000
ILC
2 pe
r col
on
Day 8Day 0
WTIl33-/-
0
2
4
6
8
%G
ATA-
3+ (IL
C)
Day 8Day 0
0
2
4
6
8
%G
ATA-
3+ (T
cel
ls)
Day 8Day 0
WTIl33-/-
*
0
2500
5000
7500
10000
T H2
per c
olon
Day 8Day 0
WTIl33-/-
*
A
B
D
CD
45
FSC-A
CD
90
CD19 C
D12
7 Lin-2
T cells
ILC
ILC WT Il33-/-
CD
90
2.3 3.8
GATA3
T cells WT Il33-/-
CD
90
GATA3
2.3 7.0
C
! 8
114!Supplementary figure 6. There is no defect in induction of anti-inflammatory and 115!epithelium healing genes or T regs in the colons of Il33-/- mice. (A-C) Quantification of 116!expression of indicated genes in the colons of WT and Il33-/- deficient mice at indicated days 117!post DSS administration by qRT-PCR. (D) Quantification of T regulatory cells gated as CD19-118!CD3+CD4+Foxp3+ cells in the lamina propria of the colon by flow cytometry. Data represent two 119!independent experiments and analyzed by Kruskal-Wallis test followed by Dunn’s post test. 120!Error bars represent mean±S.E.M with 5 mice per group per time point. 121!
122!
123!
124!
125!
126!
127!
128!
129!
130!
131!
132!
133!
134!
135!
048
300400500
Lipo
calin
Rel
ativ
e ex
pres
sion ****NS NS
Day 0 Day 8Day 4
0
2
4
6Il2
2 lo
g 10
Rel
ativ
e ex
pres
sion ****NS NS
WTIl33-/-
Day 0 Day 8Day 40
5
10
15
Reg
3βR
elat
ive
expr
essi
on NS NS NS
Day 0 Day 8Day 4
0.00.51.01.52.02.5
Occ
ludi
nR
elat
ive
expr
essi
on NS NSNS
Day 0 Day 8Day 40.0
0.5
1.0
1.5
2.0
Zo1
Rel
ativ
e ex
pres
sion **NS NS
Day 0 Day 8Day 4
0
5
10
15
Reg
3γR
elat
ive
expr
essi
on **NS NS
Day 0 Day 8Day 4
012345
Mpt
xR
elat
ive
expr
essi
on NS NSNS
Day 0 Day 8Day 40
1
2
3
4
S100
A9R
elat
ive
expr
essi
on NS NS ***
Day 0 Day 8Day 4
10
20
30
40
50
CD
4+ %
Fox
p3+ **
Day 0 Day 8Day 4
NS **
A
B
C D
! 9
136!Supplementary figure 7. Increasing Akkermansia level in WT mice increases colitis 137!susceptibility. (A) Q-PCR analysis of indicated bacteria from stool samples of WT and Il33-/- 138!mice 1-week administration of water or broad-spectrum antibiotic cocktail (125 mg/l 139!ciprofloxacin, 1 g/l bacitracin, 2 g/l streptomycin, 1.5 g/l metronidazole and 172 mg/l gentamycin) 140!in their drinking water. (B) IL-1α measurement in colon explants at day 4 post DSS 141!administration (C) Body weight loss and (D) disease activity index of mice during DSS 142!administration. (E) Colon length measurements and (F) representative colon images at day 8 143!post DSS administration. Data represent two independent experiments and analyzed by 144!Kruskal-Wallis test (A) (B) and (D) or two-way ANOVA (C) followed by Dunn’s post test. Error 145!bars represent mean±S.E.M. and each dot represents an individual mouse. N=10 mice for WT 146!and water, Il33-/- and water group, 5 for WT and antibiotics group. 147!
148! 149!
150!
151!
152!
153!
154!
155!
156!
157!
158!
159!
160!
161!
4
6
8
10
Col
on le
ngth
(cm
) ********
Water Abx
0.0
0.5
1.0
1.5
Bac
tero
ides
Rel
ativ
e to
WT
NS NS
Water Abx0.0
0.5
1.0
1.5
2.0
Pre
vote
lla R
elat
ive
to W
T
*NS
Water Abx
0 3 6 7 970
80
90
100
110
Days post DSS
Initi
al b
ody
wei
ght (
%)
Il33-/-, Water WT, Water ****
WT, Abx
0
2
4
6A
kker
man
sia
Log 10
Rel
ativ
e to
WT
********
Water Abx
WT Il33-/-
0
100
200
300
400
IL-1α
(pg/
ml)
******
Water Abx
Water WT Il33-/- WT
Abx
A B
C D E
! 10
162!Supplementary figure 8. Il33-/- littermates have decreased intestinal IgA, increased level of 163!Akkermansia and susceptibility to colitis. (A) IgA measurement by ELISA and (B) qRT-PCR 164!analysis of indicated bacteria from stool samples of WT, Il33+/- and Il33-/- mice 4 weeks after 165!separation. (C) Body weight loss of mice during DSS administration. (D) Colon length 166!measurements and (E) representative colon images at day 8 post DSS administration. Data is 167!analyzed by Kruskal-Wallis test (A) (B) and (D) or two-way ANOVA (C) followed by Dunn’s post 168!test. Error bars represent mean±S.E.M. and each dot represents an individual mouse. N=11 169!mice for WT, 14 for Il33+/- and 6 for Il33-/- group. 170!
171!
172!
173!
174!
175!
176!
177!
178!
179!
180!
181!
WT Il33+/- Il33-/-0.0
0.3
0.6
0.9
1.2In
test
inal
IgA
(uni
ts/m
g)**
*
WT Il33+/- Il33-/-0
1
2
3
4
Akkermansia
Rel
ativ
e to
WT ****NS
0 3 5 6 7 9
90
100
110
Days post DSS
Initi
al b
ody
wei
ght (
%)
WTIl33+/-
Il33-/-
** ****
WT Il33+/- Il33-/-0.0
0.5
1.0
1.5
2.0
Bacteriodes
Rel
ativ
e to
WT
NS NS
WT Il33+/- Il33-/-6
7
8
9
10
11
Col
on le
ngth
(cm
) *****NS
WT Il33+/- Il33-/-
A B
C D E
! 11
Supplementary Methods 182!
Preparation of single cell suspension from colon. 183!
Single cell suspension was prepared from the colon as described previously (1). Briefly, 184!
for removal of epithelial cells, the colon was washed, cut into small pieces, and then the 185!
pieces were incubated with calcium- and magnesium-free HBSS supplemented with 5% 186!
FBS and 5 mM EDTA (Sigma-Aldrich) at 140 rpm at 25°C for 30 min. The tissues were 187!
then incubated with RPMI 1640 containing 10% FBS and 0.5 mg/ml collagenase type IV 188!
for 1 hour at 37°C with shaking at 150 rpm. The liberated cells were collected by 189!
passage through a 70 µm nylon mesh. The isolated cells from the EDTA (epithelial) and 190!
collagenase (lamina propria) treated fractions were separated on a 40/80% 191!
discontinuous Percoll gradient (GE Bioscience). The following monoclonal antibodies 192!
were used in appropriate combinations: anti-CD3 (clone 145 – 2C11), anti-CD4 (clone 193!
RM4-5), anti-CD19 (clone 1D3), anti-IgA (clone RMA-1), anti-Foxp3 (FJK-16s), anti-194!
CD326 (clone G8.8), anti-CD16/CD32 (clone 93), anti-CD127 (clone A7R34), anti-195!
GATA-3 (clone L50-823), anti-NKp46 (clone 29A1.4), anti-CD45.2 (clone 104), anti-196!
CD90.2 (clone 30-H12), anti-MHCII (clone M5/114.152) and anti-mouse Lin cocktail 197!
(BioLegend, Catalog #133306). For intracellular cytokine staining, cells were fixed and 198!
permeabilized using fixation and permeabilization solution (eBioscience, Catalog # 00-199!
5523).For intracellular cytokine staining, cells were fixed and permeabilized using 200!
fixation and permeabilization solution (eBioscience). Intracellular staining for the Foxp3 201!
and GATA-3 transcription factors were performed using the eBioscience Foxp3 staining 202!
set according to the manufacturer’s recommendations. Flow cytometry data were 203!
acquired on LSRII (BD) and were analyzed with FlowJo software (TreeStar). 204!
! 12
Western blotting 205!
Proteins were extracted from colon tissues using RIPA lysis buffer supplemented proteinase 206!
and phosphatase inhibitors (Roche). Samples were resolved in 12-15% SDS-PAGE and 207!
transferred onto PVDF membranes. Blocking was performed in 5% milk for 1 hout and 208!
membranes were incubated in primary antibodies overnight at 4°C. Membranes were incubated 209!
with HRP-conjugated secondary antibody for 1 hour and proteins were visualized using ECL 210!
substrate (ThermoScientific). The primary antibodies were Caspase-1 p10 (1:500 dilution, sc-211!
515, Santa Cruz biotechnology) GAPDH (1:10,00 dilution, clone D16H11, Cell Signaling) and IL-212!
33 (1:1000 dilution, Catalog # AF3626 R&D systems). 213!
214!Supplementary Table 1. Real time qPCR primer sequences 215! 216!Gene Primer Sequences Gapdh Forward: 5’-CGTCCCGTAGACAAAATGGT-3’ Reverse: 5’-TTGATGGCAACAATC TCC AC-3’ β-actin Forward: 5’-GGCTGTATTCCC CTCCATCG-3’ Reverse: 5’-CCAGTTGGTAACAATGCCATG T-3’ Il1α Forward: 5’-AAAATCTCAGATTCACAACTGTTCGT-3’ Reverse: 5’-TGGCAACTCCTTCAGCAACAC-3’ Il5 Forward: 5’-GCAATGAGACGATGAGGCTT-3’ Reverse: 5’-CCCACGGACAGTTTGATTCT-3’ Il13 Forward: 5’-TGTGTCTCTCCCTCTGACCC-3’ Reverse: 5’-CACACTCCATACCATGCTGC-3’ Muc1 Forward: 5’-GCAGTCCTCAGTGGCACCTC-3’ Reverse: 5’-CACCGTGGGCTACTGGAGAG-3’ Muc2 Forward: 5’-GCTGACGAGTGGTTGGTGAATG-3’ Reverse: 5’-GATGAGGTGGCAGACAGGAGAC-3’ Muc3 Forward: 5’-CGTGGTCAACTGCGAGAATGG-3’ Reverse: 5’-CGGCTCTATCTCTACGCTCTCC-3’ Muc4 Forward: 5’-CAGCAGCCAGTGGGGACAG-3’ Reverse: 5’-CTCAGACACAGCCAGGGAACTC-3’ Il22 Forward: 5’-AGAACGTCTTCCAGGGTGAA-3’ Reverse: 5’-CAT CGA CAT AAG TCA GCA CCA G-3’ Reg3β Forward: 5’-ATGGCTCCTACTGCTATGCC-3’ Reverse: 5’-GTGTCCTCCAGGCCTCTTT-3’ Reg3γ Forward: 5’-ATGGCTCCTATTGCTATGC-3’ Reverse: 5’-GATGTCCTGAGGGCCTCTT-3’ Lcn2 Forward: 5’-ACATTTGTTCCAAGCTCCAGGGC-3’ Reverse: 5’-CATGGCGAACTGGTTGTAGTCCG-3’ Mptx Forward: 5’-CCTGTTTCTCTCTGTTCTTTCAGG-3’ Reverse: 5’-GGCCTTCATACACAGAGTGAAG-3’
! 13
Areg Forward: 5’-GCCATTATGCAGCTGCTTTGGAGC-3’ Reverse: 5’-TGTTTTTCTTGGGCTTAATCACCT-3’ S100A9 Forward: 5’-GGTGGAAGCACAGTTGGCA-3’ Reverse: 5’-GTGTCCAGGTCCTCCATGATG-3’ Occludin Forward: 5’-TTGAAAGTCCACCTCCTTACAGA -3’ Reverse: 5’-CCGGATAAAAAGAGTACGCTGG-3’ Zo1 Forward: 5’-GCCGCTAAGAGCACAGCAA-3’ Reverse: 5’-GCCCTCCTTTTAACACATCAGA -3’ Tff3 Forward: 5’-CCTGGTTGCTGGGTCCTCTG-3’ Reverse: 5’-GCCACGGTTGTTACACTGCTC-3’ pIgR Forward: 5’-AAGAACTGACCAAAGGGAGGA-3’ Reverse: 5’-AGAGTAACTTCAATTCTGCACCC-3’ Muc5ac Forward: 5’-CTGTGACATTATCCCATAAGCCC-3’ Reverse: 5’-AAGGGGTATAGCTGGCCTGA-3’ Eubacteria Forward: 5’-ACTCCTACGGGAGGCAGCAGT-3’ Reverse: 5’-ATTACCGCGGCTGCTGGC-3’ Bacteroides Forward: 5’-GGTTCTGAGAGGAGGTCCC-3’ Reverse: 5’-CTGCCTCCCGTAGGAGT-3’ Enterobacteriaceae
Forward: 5′-GTGCCAGCMG CCGCGGTAA-3′ Reverse: 5′-GCCTCAAGGG CACAACCTCC AAG-3′
γ-Proteobacteria Forward: 5’-TAACGCTTGG GAATCTGCCT RTT-3’ Reverse: 5′-CATCTRTTAG CGCCAGGCCT TGC-3′ Fungal 18s Forward: 5’- ATTGGAGGGCAAGTCTGGTG-3’ Reverse: 5′-CCGATCCCTAGTCGGCATAG-3′ Prevotellacae Forward: 5’- ATTGGAGGGCAAGTCTGGTG-3’ Reverse: 5′-CCGATCCCTAGTCGGCATAG-3′ Akkermansia Forward: 5’-CAGCACGTGAAGGTGGGGAC-3’ Reverse: 5′-CCTTGCGGTTGGCTTCAGAT-3′ Anaerostipes Forward: 5’-AAGTCGAACGAAGCACCTTG-3’ Reverse: 5′-TCCGCCACTCAGTCACAATG-3′ Dorea Forward: 5’-ACGGTACCTGACTAAGAAGCCC-3’ Reverse: 5′-CCTCAACGTCAGTCATCGTCC-3′ E. rectale Forward: 5’-ACTCCTACGGGAGGCAGC-3’ Reverse: 5′-GCTTCTTAGTCAGGTACCGTCA-3′ Flexispira Forward: 5’-AATACATGCAAGTCGAACGATGA-3’ Reverse: 5′-AATCACCGTTTCCAGTGGCT-3′ Clostridia Forward: 5’-CTCAACTTGGGTGCTGCATTT-3’ Reverse: 5′-ATTGTAGTACGTGTGTAGCCC-3′ E. coli Forward: 5’-CATGCCGCGTGTATGAAGAA-3’ Reverse: 5′-CGGGTAACGTCAATGAGCAAA-3′ Prevotella Forward: 5’-CACGGTAAACGATGGATGCC-3’ Reverse: 5′-GGTCGGGTTGCAGACC-3′ Lactobacillus Forward: 5’-GGAAACAGATGCTAATACCG-3’ Reverse: 5′-CACCGCTACACATGGAG-3′ MIB Forward: 5’-CCAGCAGCCGCGGTAATA-3’ Reverse: 5′-CGCATTCCGCATACTTCTC-3′ SFB Forward: 5’-GACGCTGAGGCATGAGAGCA-3’ Reverse: 5′-GACGGCACGGATTGTTATTC-3′ Tm7 Forward: 5’- GCAACTCTTTACGCCCAGT-3’ Reverse: 5′- GAGAGGATGATCAGCCAG-3′
! 14
!217!!218!1.! Malik!A,!Sharma!D,!St!Charles!J,!Dybas!L,!and!Mansfield!L.!Contrasting!immune!219!
responses!mediate!Campylobacter!jejuniJinduced!colitis!and!autoimmunity.!Mucosal(220!immunology.!2013.!221!
!222!
