tu anh n. pham, simon clare, david goulding, julia m ... · browne, jacqueline a. keane, andrew j....
TRANSCRIPT
Cell Host & Microbe, Volume 16
Supplemental Information
Epithelial IL-22RA1-Mediated Fucosylation Promotes Intestinal Colonization Resistance to an
Opportunistic Pathogen
Tu Anh N. Pham, Simon Clare, David Goulding, Julia M. Arasteh, Mark D. Stares, Hilary P. Browne, Jacqueline A. Keane, Andrew J. Page, Natsuhiko Kumasaka, Leanne Kane, Lynda Mottram, Katherine Harcourt, Christine Hale, Mark J. Arends, Daniel J. Gaffney, The Sanger Mouse Genetics Project, Gordon Dougan, Trevor D. Lawley
-actinP En2 SA lacZ pA pA neoR IRES
exon1 2 3 4 5 6 7
0
1
2
3
4
Tota
l cLP
cel
ls (x
106 )
A
Viability at weaning
WT
Il22ra1-/-Il22ra1+/-
0 20 40 60 80 100
MonocytesGranulocytes
CD8+ TCD4+ TT cellsB cells
WTIl22ra1-/-
% peripheral blood mononuclear cells
Bod
y w
eigh
t (g)
Age (weeks)
WT Il22ra1-/-
WT Il22ra1-/-
1
2
3
4
5
Sha
nnon
div
ersi
ty in
dex
WT Il22ra1-/-0 1000 2000 3000 4000 5000 60000
50
100
150
200
250
300
Number of sequences sampled
OTU
s
WT Il22ra1-/-
D
E
F
WT Il22ra1-/-
B
WT Il22ra1-/-0
20
40
60
80
100
Pro
potio
nal a
bund
ance
(%)
BacteroidetesFirmicutes
Actinobacteriaand Others
Proteobacteria
G
C
ns
ns
3 8 13 181015202530354045
WT femalesIl22ra1-/- femalesWT malesIl22ra1-/- males
Figure S1, related to Figure 1
Figure S1, related to Figure 1. Il22ra1-deficient mice show no phenotypic abnormalities or
signs of intestinal dysbiosis at baseline.
(A) Schematic diagram of the Il22ra1tm1a knockout-first allele with flanking Frt (blue) and loxP (pink
triangles) sites. En2 SA, mouse engrailed 2 splice acceptor; IRES, internal ribosomal entry site;
lacZ, β-galactosidase reporter; βactinR, β-actin gene promoter; neoR, neomycin resistance casette.
(B) LacZ staining of WT and Il22ra1-/- ceca, showing endogenous Il22ra1 expression.
(C) Viability at weaning of Il22ra1+/+, Il22ra1+/- and Il22ra1-/- mice (n=124) raised under SPF
conditions showing normal Mendelian ratio.
(D) Growth curves (top panel) and flow cytometric analysis of peripheral blood mononuclear
leukocytes (bottom panel) of WT and Il22ra1-/- mice (n=18-54/group).
(E) Representative H&E-stained cecal sections and total colonic lamina propria (cLP) leukocytes of
8-week old SPF WT and Il22ra1-/- mice (n=14). Scale bar, 100 µm.
(F) Rarefaction curves and Shannon diversity indices based on pyrosequencing of the 16S rRNA,
showing no significant difference in species diversity of the faecal microbiota between WT and
Il22ra1-/- mice (n=12; Mann-Whitney test).
(G) Microbiota structure of WT and Il22ra1-/- mice (n=6) showing proportional abundance of major
bacterial phyla.
Figure S2, related to Figure 1. Il22ra1-/- mice are susceptible to intestinal C. rodentium infection and DSS-colitis, but not to systemic Salmonella Typhimurium infection.WT and Il22ra1-/- mice (n=8) were infected intravenously with 105 CFU of Salmonella Typhimurium. Shown are (A) weight loss and (B) bacterial enumeration in the liver and spleen at day 14 and 28 p.i., and (C) serum anti-Salmonella titres at day 28 p.i.. Log-rank and Mann-Whitney test, *p<0.05, **p<0.001. Data are representative of at least two independent experiments.(D) Survival and weight loss of WT and Il22ra1-/- mice (n=8-12) after oral infection with 109 CFU of C. rodentium.
0 5 10 15 20 25 300
20
40
60
80
100
p = 0.0003
Il22ra1-/-WT
0 5 10 15-25-20-15-10-505
10 ***
0 5 10 15 20 25 30
-20
-10
0
10
20
Il22ra1-/-WT
Wei
ght l
oss
(%)
Days p.i.
Days p.i. Days p.i.
Sur
viva
l (%
)
Il22ra1-/-WT
Wei
ght l
oss
(%)
D
A B
WT Il22ra1 -/-0
2
4
6
8
10 ***
Path
olog
y sc
oreWT
*
Il22ra1-/-
log 10
S. T
yphi
mur
ium
CFU
LiverSpleen
Days p.i. Days p.i.
log 10
S. T
yphi
mur
ium
CFU
IgGIgG1
IgG2a100
101
102
103
104
WTIl22ra1-/-
C
14 280
1
2
3
4
5
6WTIl22ra1-/-
14 2801234567
WTIl22ra1-/-
Ant
i-Sal
mon
ella
seru
m ti
tre/m
l
E
0 2 4 6 8 10 12-25
-20
-15
-10
-5
WTIl22ra1-/-
* **p = 0.007
DSS
0 2 4 6 8 10 12 140
20
40
60
80
100
Il22ra1-/-WTS
urvi
val (
%)
Wei
ght l
oss
(%)
5
0
Days
F DSSG
Days
Figure S2, related to Figure 1
WT Il22ra1-/-0
1
2
3
4
5
Tota
l cLP
cel
ls (x
106 )
**
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
CD
8
CD4
16.7±1.3
4.9±1.0
25.3±1.8
5.4±0.5
WT Il22ra1-/-
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
103
104
105
0 102 103 104 105
0
103
104
105
TCRβ
B220
CD
25
Foxp3
15.1±1.2
12.9±1.3
WT Il22ra1 -/-
WT Il22ra1-/-6.5
±0.54.9
±0.3
0 102 103 104 105
0
102
103
104
105
0
102
103
104
105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
Ly6C
CD11b
Ly6G
WT Il22ra1-/-5.7±0.2
6.2±1.2
6.6±0.5
7.5±0.4
WT
Il22ra1-
/-WT
0
50
100
150
0
500
1000
1500
IFN
**
***
0
10
20
30
40
0
50
100
150
200
IL-1
***
0
20
40
60
0
2000
4000
6000
8000
10000
IL-6**
Tran
scrip
ts/1
00,0
00 G
apdh
Protein (pg/m
l)
Tran
scrip
ts/1
,000
Gap
dh
Protein (pg/m
l)WT WT Tr
ansc
ripts
/100
,000
Gap
dh
Protein (pg/m
l)
0
5
10
15
20
0
50
100
150
200
TNF
*****
Tran
scrip
ts/1
,000
Gap
dh
Protein (pg/m
l)
WT WTWT WT
0
20
40
60
80
0
500
1000
1500
2000
IL-17a
**
0
10
20
30
40
50
0
50
100
150
200
250
IL-17f
***
0
5
10
15
20
0
10
20
30
IL-21
*****
ND
Tran
scrip
ts/5
00,0
00 G
apdh
Protein (pg/m
l)
WT WT Tran
scrip
ts/5
00,0
00 G
apdh
Protein (pg/m
l)Tr
ansc
ripts
/500
,000
Gap
dh
Protein (pg/m
l)
WT WT WT WT0
50
100
150
1200
1400
1600
1800
2000
IL-22
*
*
Tran
scrip
ts/5
00,0
00 G
apdh
Protein (pg/m
l)
WT WT
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
Il22ra1-
/-
0
102
103
104
105
CD
4
IFN
WT Il22ra1-/-
IL-17A 0
102
103
104
105
5.7 ±0.7 15.2 ±0.9WT Il22ra1-/-
7.9 ±0.8 2.2 ±0.4
A
B D
E F
WT Il22ra1-/-0
200
400
600
800
1000
1200
WT Il22ra1-/-0
500
1000
1500
2000
Faec
al C
. rod
entiu
mes
pA Ig
A (ti
tre/g
)
Faec
al to
tal I
gA (n
g/g)
Gp = 0.06
p = 0.88
C
Figure S3, related to Figure 1
Figure S3, related to Figure 1. C. rodentium infection induces a mucosal Th1/Th17 response
in Il22ra1-/- mice. (A) Transcript levels in the cecal tissues (left y-axis) and protein levels secreted
by cLP leukocytes (right y-axis) isolated from WT and Il22ra1-/- mice at day 9 p.i. with C. rodentium,
showing Th1-associated cytokines (IFNγ, TNFα, IL-1β, IL-6) (top panels) and Th17-associated
cytokines (IL-17a, IL-17f, IL-21, IL-22) (bottom panels).
(B) Total cLP leukocytes of C. rodentium-infected WT and Il22ra1-/- mice.
Flow cytometric analysis of cLP leukocytes showing (C) CD4+ and CD8+ T cells; (D) IFNγ- and IL-
17A-expressing CD4+ T cells; (E) total T, B cells (top), and CD25+Foxp3+ regulatory T cells
(bottom panels); (F) Ly6G+CD11b+ neutrophils (top) and Ly6ChiCD11b+ monocytes (bottom
panels). Mean frequencies ± SEM of the indicated cLP populations are shown.
(G) Total (left panel) and C. rodentium-specific fecal IgA (right panel) titre of C. rodentium-infected
WT and Il22ra1-/- mice. Data are mean ± SEM from at least three independent experiments (n=3-5
each). Mann-Whitney test, p *< 0.05, **< 0.001, ***< 0.0001.
0
300000
600000
900000
1200000
1500000
1800000
2100000
2400000
2700000agg
cylI-A-B-M-S-L-R2-R1
sprEgelE
ace
ef0093
ef2505
bsh
cob
cadccf
kpr
gls24
nanE
ef1020
2.93 MbE. faecalis
0
500
1000
1500 *
Ser
um IL
-6 (p
g/m
l)i.p
Citro
i.p m
EF 0
50
100
150
200 *
Ser
um T
NF
(pg/
ml)
i.p C
itro
i.p m
EF
E
AW
eigh
t los
s (%
)
0 2 4 6 8 10 12 14-25
-20-15
-10
-5
0
5
10
Days p.i.
+Amp
B C
WT
C. r
oden
tium
CFU
/g fe
ces
0 2 4 6 8 10 12 14 16 18 20
100
102
104
106
108
1010
Days p.i.
+Amp
WT
*
0 2 4 6 8 10 12 14 16100102
104105106107108109
Days p.i.
Ent
eroc
occu
s C
FU/g
fece
s +Amp
WT
*
D
Il22ra1-/- + PBSIl22ra1-/- + Amp
Il22ra1-/- + PBSIl22ra1-/- + Amp
Il22ra1-/- + PBSIl22ra1-/- + Amp
Figure S4, related to Figure 2
Figure S4, related to Figure 2. A murine Enterococcus faecalis isolate harbors multiple
virulence genes and shows pathogenic potential.
(A) Serum levels of TNFα and IL-6 of groups of WT mice (n=6-10) following intraperitoneal infection
with either C. rodentium (‘i.p Citro’) or E. faecalis (‘i.p mEF’), in two independent experiments.
(B) Weight loss; (C) fecal shedding of enterococci and of (D) C. rodentium by C. rodentium-infected
WT and Il22ra1-/- mice treated i.p. with ampicillin or PBS controls. Data are from two independent
experiments (n=4-6 each). Grey area represents limit of detection.
(E) Genomic annotation of the murine E. faecalis isolate from Il22ra1-/- mice (See also Table S3),
based on the genome of V583 (Paulsen et al., 2003) - a vancomycin-resistant sepsis strain, and the
pathogenicity island of the nosocomial MMH594 strain (Shankar et al., 2002). From outside to
inside: track 1, predicted coding sequences (CDS) on the forward strand; track 2, CDS on the
reverse strand. CDS features are coloured according to functional categories: grey, virulence genes
identified by alignment with the E. faecalis MMH594 pathogenicity island; purple, cell wall/surface
adhesion genes; yellow, IS elements; blue, antibiotic resistance genes; orange, pheromone
lipoproteins; light cyan, remaining CDS features with translated nucleotide sequence homologous
to those within the V583 genome; pink, non-homologous CDS features. Track 3, nucleotide BLAST
hits > 1,000-bp long with > 90% identity to the V583 genome, at p-value cut-off < 0.001. Track 4,
ribosomal RNAs (blue) and transfer RNAs (green). Track 5 and 6, GC content and GC skew
(GC/(G+C)), coloured green (above average) or magenta (below average). Virulence genes labeled
are (clockwise from origin): gls24, general stress protein; ef0093, ef2505, cell wall surface adhesion
proteins; nanE, N-acetylmannosamine-6-phosphate epimerase; cylI-A-B-M-S-L-R2-R1, cytolysin
operon; agg, aggregation substance; ef1020, glycosyl hydrolase; ace, E. faecalis adhesin; kpr,
ketopantoate reductase; sprE, serine protease; gelE, gelatinase/coccolysin; cob, cad, ccf, sex
pheromones; bsh, bile salt hydrolase. Multiple mobile elements flanking the cytolysin operon within
a region of low G+C content (32.2% versus 37.3% in the entire genome) are found.
Day 0
Day 9
WT2
WT1
KO2KO3KO1
WT2 WT3WT1
KO2KO3
KO1
0 2000 4000 60000
50
100
150
200
0 2000 4000 60000
100
200
300
OTU
sO
TUs
WT3
Number of sequences sampled
Number of sequences sampled
Enterob
acter
iacea
e
Erysipe
lotric
hace
ae
Lach
nosp
irace
ae
Lacto
bacill
acea
e
Rikene
llace
ae
Rumino
cocc
acea
e
Suttere
llace
ae
0
1
2
3
4***
***
*
*
*****
***
******
**
** *
***Lo
g 10 A
bund
ance
1 2 1 2 1 2 1 2 1 2 1 2 1 2Family
Faecal microbiota (day 9 p.i.)Il22ra1-/-WT
A B
0
1
2
3
Log 1
0 Abu
ndan
ce
Enterob
acter
iacea
e
Erysipe
lotric
hace
ae
Lach
nosp
irace
ae
Lacto
bacill
acea
e
Rikene
llace
ae
Rumino
cocc
acea
e
Suttere
llace
aeFamily
Il22ra1-/-WT
* nsns
ns
ns
ns
ns
Faecal microbiota (day 0 p.i.)
056789
1011
** ***
Log 1
0 C. r
oden
tium
CFU
/g
luminal luminal mucosalinfectednaive
WTIl22ra1 -/-
02345678 * **
luminal luminal mucosalinfectednaive
WTIl22ra1 -/-
Log 1
0 Ent
eroc
occu
s C
FU/g
naive infected123456789
WT
ns
***
log
(16S
rRN
A c
opie
s /g
)
10 Il22ra1 -/-
Faecal Enterobacteriaceae
naive infected
9
12345678
WT
ns
log
(16S
rRN
A c
opie
s /g
)
Il22ra1 -/-
Faecal Enterococcus spp.
***
luminal luminal mucosal
10
123456789
ns
*****
infected
log
(16S
rRN
A c
opie
s /g
)
naive
Caecal EnterobacteriaceaeWTIl22ra1 -/-
mucosal
*
luminal luminal
10
123456789
ns
***lo
g (1
6S rR
NA
cop
ies
/g)
naive infected
Caecal Enterococcus spp.WTIl22ra1 -/-
02456789
1011 * *
ns
WTIl22ra1 -/-
luminal luminal mucosalinfectednaive
Log 1
0 Ent
eroc
occu
s C
FU/g 4
78
3
63
4572
71
9
6087
17
89
Day 0 Day 7 Cecal mucosa
WT(n = 8)
Il22ra1-/-(n = 8)
C
D
E F
Figure S5, related to Figure 3
Figure S5, related to Figure 3. Il22ra1 deficiency leads to severe dysbiosis, loss of health-
associated bacterial groups, and expansion of E. faecalis during intestinal inflammation.
(A) Rarefaction curves of 16S rRNA gene pyrosequences from the microbiota of WT and Il22ra1-/-
mice (n = 3) before (day 0) and at the peak of C. rodentium infection (day 9 p.i.), showing loss of
diversity during infection.
(B) Fecal microbiota of Il22ra1-/- mice compared to WT equivalents at day 0 and day 9 p.i with C.
rodentium, showing consistent bloom of Enterobacteriaceae and depletion of health-associated
commensals (Lactobacillaceae, Rikenellaceae and Ruminococcaceae families). Significant
changes at the taxonomic Family level of OTUs (97% identity) were identified by 16S
pyrosequencing of samples from two independent experiments (each with 3 mice/group), denoted 1
and 2. P-values * < 0.001, ** < 0.00001, *** < 0.0000001.
(C) qPCR of Enterobacteriaceae (left panels) and Enterococcus spp. (right panels) in the fecal and
cecal microbiota of naive and infected WT and Il22ra1-/- mice.
(D) Enumeration of C. rodentium and enterococci in the cecal lumen and associated with the
mucosa of naïve and infected WT and Il22ra1-/- mice, determined by selective plating. Data in ©
and (D) are from three independent infections (n=6-12). ns, not significant; ***p < 0.0001; **p <
0.001.
(E) CFU of Enterococcus spp. as determined by selective plating, and (F) 16S rRNA sequencing
profile of enterococci in the feces and cecal mucosa of WT and Il22ra1-/- littermates treated with
DSS (n=8) in 2 independent experiments, showing preferential expansion of E. faecalis (red)
relative to other intestinal enterococci (E. gallinarum, grey) during DSS-induced colitis. Shown are
numbers of Enterococcus sequences matched to a species ID.
Il22ra1 -/-
G
WT
G
Mean normalised expression
row min row max
RegIIIγ regenerating islet-derived 3 gammaKOxC
trl
KOxIL-22
WTxC
trl
WTxIL
-22
Infla
mm
ator
y/de
fens
e re
spon
se/
resp
onse
to w
ound
ing
Gly
copr
otei
n m
etab
olic
pro
cess
Ret
inoi
c ac
idm
etab
olis
mR
espo
nse
to
oxid
ativ
e st
ress
Dru
g m
etab
olis
m/
Oxi
datio
n re
duct
ion
DN
A re
plic
atio
n/C
ell c
ycle
RegIIIβ regenerating islet-derived 3 betaChi2l4 chitinase-3 like 4Saa2 serum amyloid A2Cxcl5 chemokine (C-X-C motif) ligand 5Tmem73 transmembrane protein 173; StingEfemp2 EGF-containing fibulin-like extracellular matrix protein 2Saa1 serum amyloid A1Nos2 nitric oxide synthase 2, inducibleCxcl3 chemokine (C-X-C motif) ligand 3Chst4 carbohydrate (chondroitin 6/keratan) sulfotransferase 4 Tac1 tachykinin 1 Clec2h C-type lectin domain family 2, member h C3 complement component 3 Pros1 protein S (alpha) Cxcl1 chemokine (C-X-C motif) ligand 1Nupr1 nuclear protein 1 Bnip3 BCL2/adenovirus E1B interacting protein 3 Nfkbiz nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, zeta Sec1 secretory blood group 1 Fut2 fucosyltransferase 2 B3gnt7 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 7 B3galt5 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 5 Unc13a unc-13 homolog A (C. elegans) St8sia5 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 Acan aggrecan
Rdh9 retinol dehydrogenase 9 Rdh16 retinol dehydrogenase 16Dhrs9 dehydrogenase/reductase (SDR family) member 9 Aldh1a3 aldehyde dehydrogenase family 1, subfamily A3 Lpo lactoperoxidase Duox2 dual oxidase 2Duox1 dual oxidase 1Tat tyrosine aminotransferase
Cyp2c67 cytochrome P450, family 2, subfamily c, polypeptide 67 Cyp2c69 cytochrome P450, family 2, subfamily c, polypeptide 69Cyp2c40 cytochrome P450, family 2, subfamily c, polypeptide 40Cyp2c68 cytochrome P450, family 2, subfamily c, polypeptide 68Ugt2b36 UDP glucuronosyltransferase 2 family, polypeptide B36 Ugt2b5 UDP glucuronosyltransferase 2 family, polypeptide B5Cyp2c55 cytochrome P450, family 2, subfamily c, polypeptide 55 Cyp4b1 cytochrome P450, family 4, subfamily b, polypeptide 1 Hao2 hydroxyacid oxidase 2 Nox1 NADPH oxidase 1 Cyp1a1 cytochrome P450, family 1, subfamily a, polypeptide 1 Hsd17b14 hydroxysteroid (17-beta) dehydrogenase 14 Hpgd hydroxyprostaglandin dehydrogenase 15 (NAD) Abp1 amiloride binding protein 1 (amine oxidase, copper-containing) Cyp2j9 cytochrome P450, family 2, subfamily j, polypeptide 9 Akr1c14 aldo-keto reductase family 1, member C19 Gstm3 glutathione S-transferase, mu 3 Gsta3 glutathione S-transferase, alpha 3 Gstm1 glutathione S-transferase, mu 1Cyp2d26 cytochrome P450, family 2, subfamily d, polypeptide 26 Hsd3b2 hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 Rec8 REC8 homolog (yeast) Myb myeloblastosis oncogene
Il22ra1 genotype x IL-22 treatmentinteraction term
-3.8 -0.6
Tmem173
Reg3g
Ifn
Oas2Ifit1 Caecam1/2
Plac8
IL-12Ly6 family
Ifng
RelJakReg3b
Zbp1
Ncoa7
Socs3
TifaItgb8
Aldh1a3
Tnfaip8Tat
Nfkbiz
Cd38
Slpi
NFkB
Dmbt1
WT WT WT WTIl22 -/- Il22 -/- Il22 -/- Il22 -/-
Ctrl+ IL-22 (50 ng/ml)
Reg3 Reg3 S100a8 S100a9
Rel
ativ
e m
RN
A le
vels
per G
apdh
0.1
1
10
100
1000
WT Il22ra1-/- WT WT WT0.1
1
10
100
1000
Reg3 Reg3 S100a8 S100a9
********* ***
Il22ra1-/- Il22ra1-/- Il22ra1-/-Rel
ativ
e m
RN
A le
vels
per G
apdh
Ctrl+ IL-22 (50 ng/ml)
C D
E
F
Reg
IIIγ
trans
crip
ts/1
0,00
0 G
apdh
naive infected
WT W
T
Il22ra
1-/-
0
250
500
750
1000 ***
A
spleen liver
0.0
0.5
1.0
1.5
2.0
Org
an w
eigh
t (g)
WT
RegIIIγ
-/-W
T
RegIIIγ
-/-
B ns
ns
Figure S6, related to Figures 4 and 5
Il22ra
1-/-
Figure S6, related to Figures 4 and 5. Global transcriptomics of IL-22RA1 signaling in
colonic epithelial organoids reveal antimicrobial and glycosylation processes beyond
RegIIIγ.
(A) qRT-PCR of RegIIIγ expression in the cecal tissues of naive and C. rodentium-infected WT and
Il22ra1-/- mice (n =12-16/group), ***p<0.0001.
(B) Liver and spleen weights of C. rodentium-infected WT and RegIIIγ-/- mice at day 9 p.i., showing
no significant difference. No bacterial dissemination was detected by non-selective culturing.
(C) Representative toluidine blue-stained semithin section of WT and Il22ra1-/- organoids showing
columnar enterocytes and mucus-producing goblet cells (‘G’). Scale bar, 10 µm.
(D) WT, Il22ra1-/- and Il22-/- organoids were stimulated with recombinant mouse IL-22 and levels of
RegIIIγ, RegIIIβ, S100a8, S100a9 transcripts were measured by qRT-PCR. Results from biological
and technical duplicates. Two-way ANOVA, p-value ***<0.0001.
(E) Heatmap showing relative mean expression of genes significantly induced or down-regulated by
IL-22, associated with key Gene Ontology biological processes in Figure 5.
(F) Top network with “antimicrobial function”, identified by Ingenuity Pathway analysis among genes
significantly induced by IL-22 in wildtype organoids (p < 10-12). Nodes (i.e. genes) are colored
according to their magnitude of expression changes.
Table S1, related to Figure 1. Isolation and identification by 16S rRNA gene sequencing of systemic bacterial isolates in C. rodentium-infected WT and Il22ra1-/- mice.
Il22ra1-/- WT Day 5 p.i. Day 9-10 p.i. Subtotal Subtotal Taxonomy A B C D E F G H I J K (n=28) A C K (n=10)
Phylum Family Species*
Number of isolates sequenced 28 44 39 50 41 68 58 97 88 103 221 726 32 25 23 80 Proteobacteria Enterobacteriaceae Citrobacter
rodentium 17 28 20 16 14 10 27 22 31 72 163 355 20 4 3 27
Firmicutes Enterococcaceae Enterococcus faecalis 10 14 19 22 25 48 8 44 49 16 31 243 0 0 0 0
Other Firmicutes Staphylococcaceae Staphylococcus lentus 0 0 0 0 0 3 1 0 1 0 1 6 0 0 0 0
Staphylococcus napelensis 0 0 0 0 0 0 5 0 0 1 3 9 0 0 0 0
Staphylococcus epidermidis 0 0 0 0 0 0 1 1 0 0 0 2 0 0 0 0
Lactobacillaceae Lactobacillus murinus 1 2 0 4 0 5 6 6 1 4 0 26 4 8 12 24
Lactobacillus johnsonii 0 0 0 4 0 0 1 1 0 0 0 6 0 2 0 2
Lactobacillus reuteri 0 0 0 1 0 0 0 2 0 0 0 3 0 2 0 2
Lactobacillus intestinalis 0 0 0 0 0 0 1 0 0 0 1 2 0 0 0 0
Bacteroidetes Porphyromonodaceae Parabacteroides distasonis 0 0 0 0 0 2 1 1 0 3 5 13 4 0 0 4
Barnesiella intestinihominis 0 0 0 3 1 0 2 0 0 0 10 16 0 0 2 2
Bacteroidaceae Bacteroides uniformis 0 0 0 0 0 0 2 10 0 3 0 13 0 0 0 0
Bacteroides acidifaciens 0 0 0 0 1 0 3 5 0 4 5 17 4 5 0 9
Bacteroides xylanisolvens 0 0 0 0 0 0 2 3 0 0 2 7 0 4 0 4
Other Bacteria** 0 0 0 0 0 0 0 2 6 0 0 8 0 0 6 6
A-K indicate independent experiments with similar outcome performed over the course of 18 months at our animal facility. In each experiment, Il22ra1-/- animals showing significant signs of morbidity were examined for bacterial breakthrough in the livers and spleens. Isolates from non-selective culture media were profiled by 16S rRNA gene sequencing. *Species: the closest match by 16S rRNA gene sequence identity (> 97%). **Other Bacteria indicate rare bacterial isolates only detected in one experiment but not others, and include Parasutterella excrementihominis, Bifidobacterium longum, Adlercreutzia spp., Alistipes spp. and Micrococcus spp.
Table S2, related to Figure 1. Isolation and identification by 16S rRNA gene sequencing of systemic bacterial isolates in DSS-treated WT and Il22ra1-/- littermates.
Taxonomy WT Il22ra1-/-
Phylum Family Species*
Number of isolates sequenced 84 247 Proteobacteria Enterobacteriaceae Escherichia coli 21 56 Firmicutes Enterococcaceae Enterococcus faecalis 2 83 Enterococcus gallinarum 1 0 Other Firmicutes Staphylococcaceae Staphylococcus lentus 3 6 Staphylococcus napelensis 3 12 Staphylococcus hemolyticus 2 0 Staphylococcus epidermidis 0 2 Lactobacillaceae Lactobacillus murinus 17 48 Lactobacillus johnsonii 3 7 Lactobacillus reuteri 0 12 Lactobacillus intestinalis 1 1 Bacteroidetes Porphyromonodaceae Parabacteroides distasonis 10 7 Barnesiella intestinihomonis 1 0 Bacteroidaceae Bacteroides uniformis 10 8 Bacteroides acidifaciens 2 1 Bacteroides xylanisolvens 2 3 Actinobacteria Coriobacteriaceae Adlercreutzia equolifaciens 6 1
Shown are results from two independent experiments (n=8-10) with similar outcome. Isolates from non-selective culture media were profiled by 16S rRNA gene sequencing. *Species: the closest match by 16S rRNA gene sequence identity (> 97%).
Table S3, related to Figure 2. List of virulence factors identified in the genome of a pathogenic murine Enterococcus faecalis isolate. Virulence genes Cytolysin operon (cylR1-R2-L-S-M-B-A-I)
Production and activation of cytolysin capable of lysing erythrocytes, inflammatory cells and Gram-positive bacteria; contributes to virulence and mortality in murine peritonitis and endocarditis models (Chow et al., 1993; Ike et al., 1984; Jett et al., 1992).
Aggregation substance (agg) Important for adherence and invasion of enterocytes in vitro; plays a role in murine urinary tract infection and endocarditis; facilitates bacterial clumping and survival in neutrophils (Chow et al., 1993; Rakita et al., 1999).
Gelatinase/coccolysin (gelE) Zinc-dependent metalloprotease important for mortality in murine peritonitis (Engelbert et al., 2004; Singh et al., 1998).
Serine protease (sprE) Exoenzyme involved in pathogenesis in murine peritonitis and rabbit endophthalmitis models (Engelbert et al., 2004; Singh et al., 1998).
Additional factors relevant to pathogenesis Enterococcus faecalis adhesin (ace)
Microbial surface component recognising adhesive matrix molecule (MSCRAMM) involved in host cell adherence and endocarditis (Nallapareddy et al., 2000).
Cell wall surface anchor proteins (ef0093, ef2505)
Putative adherence factors that may facilitate binding to host extracellular matrix.
Bile salt hydrolase (bsh) Defense against toxicity of bile salts, increased intestinal colonisation and systemic lethality in Listeria monocytogenes infection (Dussurget et al., 2002).
Sex pheromones (cob, cad, ccf)
Chemoattractant for neutrophils and modulation of host inflammation (Sannomiya et al., 1990).
Gls24 protein (gls24) General stress response protein induced upon nutrient starvation and exposure to heavy metals and bile salts (Giard et al., 2000).
N-acetylmannosamine-6-phosphate epimerase (nanE) Glycosyl hydrolase (ef1020) Ketopantoate reductase (kpr)
Carbohydrate and coenzyme A metabolism. May contribute to survival in the nutritionally depleted environment of the gastrointestinal tract.
Supplemental Table 4, related to Figure 2. Comparative genomics of E. faecalis isolates from various nosocomial, commensal and animal origins, and isolates from different mice colonies. (separate .xls file) Supplemental Table 5, related to Figure 5. RNAseq of WT and Il22ra1-/- epithelial organoids reveals diverse antimicrobial and glycosylation genes and pathways. (separate .xls file)
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Mice Experiments
C57BL/6N and Il22ra1-/- mice were obtained from inbred colonies from the Sanger Mouse Genetics
Project (Bradley et al., 2012; White et al., 2013). Il22-/- mice, as previously described (Kreymborg et
al., 2007), were received from Prof. Fiona Powrie (University of Oxford). Both WT and Il22ra1-/-
derived from separate colonies and littermates derived from heterozygote breeding pairs have been
used for infection and DSS experiments, with similar outcomes. Il22ra1-/- littermates were also used
for experiments involving ampicillin or 2’-fucosyllactose treatment. In experiments to profile the
enterococcal community dynamics during inflammation, mice were singly housed from Day 0 post-
infection or DSS initiation to minimize transfer of bacteria by coprophagia. Mice from the same litters
were routinely kept together prior to infection or DSS administration, and were fed a regular
autoclaved chow diet (LabDiet) ad libitum. All mice were matched for age (6-9 weeks) and gender
within each experiment and infected mice were monitored daily for weight loss.
Infection challenges were performed by oral gavage with 109 CFU of C. rodentium (KanR, NalR)
(Wiles et al., 2006) or i.v. injection of 2 x 105 CFU of Salmonella enterica serovar Typhimurium M525
TETc (Clare et al., 2003). Bacterial colonization in the liver, spleen, cecal contents and intestinal
mucosa was determined by serial dilution plating of homogenized tissues on antibiotic-
supplemented agar. Serum IL-6 and TNFα were determined with the Mouse IL-6 and TNFα Ready-
Set-Go! ELISA kits (eBioscience). To test for an attenuating effect on susceptibility to infection,
Il22ra1-/- mice were administered i.p. 0.1 mg ampicillin (Sigma) in 0.1 ml PBS at Day 5, 7 and 9 p.i.,
or oral 2 mg 2’-fucosyllactose (Carbosynth Ltd) in 0.1 ml PBS at Day 5, 6, 7 and 8 p.i. Virulence
testing of E. faecalis was conducted by administering i.p. 109 CFU of the recovered murine E.
faecalis strain, using the peritonitis model as previously described (Bourgogne et al., 2008; Singh et
al., 1998).
Histology
To evaluate disease pathology, we fixed cecal segments in 4% paraformaldehyde and stained 5 µm-
thick paraffin sections in hematoxylin and eosin according to standard protocol. Scoring of intestinal
inflammation was performed in a blinded manner by a clinical pathologist as follow: Submucosal
edema: mild-1, moderate-2, severe-3; submucosal inflammation: mild-1, moderate-2, severe-3;
mucosal inflammation: mild-1, moderate-2, severe-3; crypt abscess: absent-0, present-1. For
immunofluorescence, we stained 5 µm-thick frozen sections of mouse cecal tissues with α-
Citrobacter translocated intimin receptor antibody (Tir) (1/1000), or α-Enterococcus spp. antibody
(1/200, LSBio). Sections were mounted using ProLong Gold antifade reagent (Molecular Probes)
containing 4’,6’-diamidino-2-phenylindole (DAPI) for nuclear staining. Light and fluorescence
microscopy images were taken with a Zeiss Axiovert 200M instrument using the AxioVision
software.
Quantitative Real-Time PCR
The terminal 3-mm of the ceca were excised and preserved in RNALater (QIAGEN) for subsequent
total RNA extraction. Expression of mouse genes in the cecal tissues were assessed using the
following TaqMan primers and probes. qPCR conditions were 95oC for 15 min, 40 cycles of 95oC for
15 sec and 60oC for 1 min. mRNA levels were normalized relative to Gapdh.
Gapdh Forward (F): TGTGTCCGTCGTGGATCTGA Reverse (R): CACCACCTTCTTGATGTCATCATAC Probe (Pr): TGCCGCCTGGAGAAACCTGCC
TNF-α F: CATCTTCTCAAAATTCGAGTGACAA R: CCAGCTGCTCCTCCACTTG Pr: CCTGTAGCCCACGTCGTAGCAAACCA
IFN-γ F: CAGCAACAGCAAGGCGAAA R: CTGGACCTGTGGGTTGTTGAC Pr: AGGATGCATTCATGAGTATTGCCAAGTTTGA
IL-1β F: CCAAAAGATGAAGGGCTGCTT R: TGCTGCTGCGAGATTTGAAG Pr: CAAACCTTTGACCTGGGCTGTCCTGA
IL-6 F: ACAAGTCGGAGGCTTAATTACACAT R: TTGCCATTGCACAACTCTTTTC Pr: TTCTCTGGGAAATCGTGGAAATG
IL-22 F: TCCGAGGAGTCAGTGCTAA R: AGAACGTCTTCCAGGGTGAA Pr: TGAGCACCTGCTTCATCAGGTAGCA
IL-17a F: CACCTCACACGAGGCACAAG R: GCAGCAACAGCATCAGAGACA
Pr: ACCCAGCACCAGCTGATCAGGACG IL-17f F: CCATTGGAGAAACCAGCATGA
R: CCCAACATCAACAGTAGCAAAGAC Pr: TGCACCCGTGAAACAGCCATGG
IL-21 F: GGACAGTGGCCCATAAATCAA R: GTTCAGGATCCAAGTCATTTTCATAG Pr: CCCCAAGGGCCAGATCGCCT
Reg3β F: ATGGCTCCTACTGCTATGCC R: GTGTCCTCCAGGCCTCTTT Pr: TGATGCAGAACTGGCCTGCCA
Reg3γ F: ATGGCTCCTATTGCTATGCC R: GATGTCCTGAGGGCCTCTT Pr: TGGCAGGCCATATCTGCATCATACC
S100a8 F: TGTCCTCAGTTTGTGCAGAATATAAA R: TCACCATCGCAAGGAACTCC Pr: CGAAAACTTGTTCAGAGAATTGGACATCAATAGTGA
S100a9 F: GGTGGAAGCACAGTTGGCA R: GTGTCCAGGTCCTCCATGATG Pr: TGAAGAAAGAGAAGAGAAATGAAGCCCTCATAAATG
Lcn2 F: GGCCAGTTCACTCTGGGAAA R: CCACTTGCACATTGTAGCTCTGT Pr: ATGCACAGGTATCCTCAGG
mCramp F: GCCGCTGATTCTTTTGACATC R: GCCAGCCGGGAAATTTTCT Pr: AACGAGCCTGGTGCACAGCCCT
Fut2 F: TGCACTGGCCAGGATGAA R: GCGCTAGAGCGTTGTGCAT Pr: TCGGCTTGCCTTCATCCCTGAATC
Flow Cytometry
To reduce non-specific Fc receptor-binding, cells were pre-incubated with anti-CD16/CD32 (1/100,
clone 2.4G2, BD Biosciences) for 15 min at 4oC. Cell viability was determined by a further 15 min
incubation in Fixable Viability Dye eFluor780 (1/1000 in PBS, eBioscience). We used a combination
of the following antibodies to label surface or intracellular antigens on cLP cells: α-CD45 (clone 30-
F11), CD3 (17A2), CD11b (M1/70), CD11c (N418), Ly6G (1A8), CD103 (M290), CD64 (X54-5/7.1),
CD115 (AFS98) (from BD Biosciences), CD4 (GK1.5), CD8a (53-6.7), CD25 (PC61.5), Foxp3 (FJK-
16S), MHC class II (M5/114.15.2) (from eBiosciences), and Ly6C (HK1.4; Biolegend). To detect
intracellular cytokines, we stimulated 5 x 105 cLP cells for 4-5 hours in RPMI containing 50 ng/ml
phorbol myristate acetate (PMA, Sigma) and 0.5 µg/ml ionomycin (Sigma) in the presence of
GolgiStop solution with Brefeldin A (1 µl/ml, BD Biosciences). Cells were then fixed and
permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences), and stained for 1 hour with α-IFNγ
(clone XMG1.2) and IL-17A (TC11-18H10; both from BD Biosciences). Flow cytometry data were
acquired on an LSRFortessa instrument (BD Biosciences) and analysed using FlowJo software
(TreeStar).
Genomic sequencing and comparative genomics of Enterococcus faecalis
Genomic DNA from Enterococcus faecalis isolates was prepared from 24-hour cultures using
standard phenol-chloroform extraction procedure (He et al., 2010). We created multiplexed libraries
with 150-bp paired-end reads and sequenced the genomes on a MiSeq 2000 platform (Illumina).
Contigs were assembled de novo using Velvet (Zerbino and Birney, 2008), and aligned with the
V583 E. faecalis genome (NCBI accession number NC_004668) using the MUMmer package
(nucmer programme) (Kurtz et al., 2004) implemented in ABACAS (Assefa et al., 2009). We
performed gene prediction using Prodigal (Hyatt et al., 2010), followed by annotation transfer using
data from the V583 genome and the MMH594 pathogenicity island (accession number AF454824).
Further manual annotation and sequence alignment were done using Artemis (Rutherford et al.,
2000) and ACT (Carver et al., 2005). Circular representation of the murine E. faecalis genome was
done using DNAPlotter 4.1 (Carver et al., 2009). Based on the previously published full and draft
genomes of diverse E. faecalis strains from animal and human sources (Arias et al., 2011; Brede et
al., 2011; Fritzenwanker et al., 2013; McBride et al., 2007; Ruiz-Garbajosa et al., 2006; Zischka et
al., 2012) (Table S4), we constructed maximum likelihood dendogram with Seaview (Gouy et al.,
2010), using the concatenated sequences of E. faecalis multilocus sequencing typing genes (aroD,
gdh, gki, gyd, pstS, xpt, yqil).
To profile the diversity of enterococci in the mouse microbiota, we picked at least 10 identical
colonies of enterococci grown on selective Bile-Esculin-Azide agar per sample per time point for 16S
rRNA gene sequencing. Whole-genome sequencing was performed with 30 representative
Enterococcus faecalis isolates from different mouse colonies using Illumina MiSeq, generating
approximately 50X genome coverage per isolate (Table S5). The genomes of isolates from different
mouse colonies were compared to identify any genetic variations (e.g. single nucleotide
polymorphisms (SNPs) or insertions/deletions).
Microbiota Analysis
To study the temporal changes of C. rodentium infection on the microbial ecology of wild-type and
Il22ra1-/- mice, we collected faecal pellets of individual earmarked animals, naïve and infected (n =
3/group) before inoculation and again after 9 days, at which point mice were culled to obtain the
luminal contents of the caecum by manual extrusion. Faecal samples were similarly obtained from
Il22ra1-/- mice infected with C. rodentium, treated with 2’-fucosyllactose or PBS control. To extract
total DNA, we used the FastDNA Spin Kit for Soil with additional use of the FastPrep Instrument (MP
Biomedicals) to aid sample lysis. V5-V3 regions of bacterial 16S rRNA genes were PCR amplified
with the high-fidelity AccuPrime Taq Polymerase (Invitrogen) and the following primers: 338F, 5’-
CCGTCAATTCMTTTRAGT - 3’; 926R, 5’ - ACTCCTACGGGAGGCAGCAG - 3’. PCR products were
quantified with a Qubit 2.0 Fluorometer (Invitrogen) and equimolar volumes of each sample were
pyrosequenced on a 454 GS-FLX Titanium platform. Quality filtering was applied to include only
high quality (minimum average quality score of 35 in a 50-bp sliding window), non-chimeric
sequences containing the exact 4-nt tags, without homopolymer or ambiguous nucleotide. A mock
community sample consisting of genomic DNA from the following bacterial species: Parabacteroides
distasonis, Bacteroides thetaiotaomicron, Escherichia coli, Adlercreutzia equolifaciens, Clostridium
paraputrificum, Enterococcus faecalis was included to assess sequencing errors after quality filtering
(Schloss et al., 2011). Each datasets contained at least 600,000 sequences (approximately 4000
reads/sample post-quality filtering). Following alignment with the SILVA-derived reference database
(Pruesse et al., 2007) using the Mothur software package (Schloss et al., 2009), we generated
operational taxonomic units (OTUs) from unique sequences at 3% distance and assigned the
taxonomic classifications from phyla to genus level according to the Ribosomal Database Project
(Cole et al., 2009) and SILVA databases. Rarefaction analyses, calculations of alpha diversity
indices, and community clustering analyses were implemented using the Mothur software.
Intestinal Organoids Culture
Colonic organoids were isolated and cultured in vitro as described (Sato et al., 2011; Yui et al.,
2012). In brief, mouse colon segments were opened longitudinally, washed and incubated with
agitation in 2mM EDTA in PBS for 35 min at 4oC to dissociate the epithelial cells. Colonic crypts
were obtained by vigorous resuspension of tissue fragments with a 5-mL pipette in sedimentation
buffer (DMEM (Sigma) supplemented with 2mM L-glutamine (Invitrogen), 5% FBS (Sigma) and 2%
(w/v) D-sorbitol (Sigma)). We collected the supernatant containing crypts, and pelleted them by
centrifugation at 200-300g for 3 min. The pellet were washed five times in sedimentation buffer to
further remove non-epithelial cells, and resuspended in basal growth medium (advanced DMEM/F12
(Sigma) supplemented with 2mM L-glutamine, 5mM HEPES, 1 X N2 and B27 media supplements,
pencillin/streptomycin (all from Invitrogen), 1mM N-acetylcysteine and 5% FBS (Sigma)). About 500-
1000 crypt units were mixed with 100 µl phenol-red free Matrigel (Invitrogen) and allowed to solidify
in a 24-well plate. We then added 1ml/well of basal growth medium supplemented with 100 ng/ml
recombinant mouse Wnt-3a (Peprotech), 1 µg/ml recombinant mouse R-Spondin 1 (R&D Systems),
100 ng/ml Noggin (Peprotech) and 50 ng/ml EGF (Invitrogen) and replaced the medium every 2
days. Light microscopy was performed after 6-7 days of culture. For transmission electron
microscopy, organoids were fixed in 2.5% glutaraldehyde/2% paraformaldehyde, post-fixed with
0.1M sodium cacodylate buffer and 1% osmium tetroxide, followed by dehydration through an
ethanol series and embedding in TAAB 812 resin. We contrasted ultrathin sections with uranyl
acetate and lead nitrate, and recorded images on an FEI 120kV Spirit Biotwin microscope.
RNA Sequencing and Analysis
We performed RNA sequencing on 5-day old organoids from wild-type and Il22ra1-/- mice
(n=4/genotype) in technical duplicates. Organoids were either kept in complete growth medium or
stimulated with 50 ng/ml mouse recombinant IL-22 (Cambridge Bioscience) for 16 hours. After
adding ice-cold Cell disaggregation medium (Invitrogen) for 15 minutes to dissolve Matrigel, we
extracted total RNA using the RNeasy Micro kit (Qiagen), including a 10-minute treatment with
RNAase-free DNAse I (Qiagen) to minimise contaminating genomic DNA. We generated multiplexed
cDNA libraries from high-quality RNA samples (RNA integrity number ≥ 7.0) according to the
Illumina TruSeq RNA Preparation protocol, and sequenced them on an Illumina HiSeq platform.
Each lane of Illumina sequence was assessed for quality based on GC content, average base
quality and adapter contamination. We obtained between 4.0 and 10.9 Gbp of RNA-seq data per
sample with paired-end reads 76-bp in length. RNA-seq reads were aligned to the mm10/NCBIM37
reference genome with BWA 0.6.1 (Li and Durbin, 2010). Gene expression values were computed
from the read alignments to the coding sequence to generate the number of reads mapping and
RPKM (reads per kilobase per million). We next used the whole-genome read count data to ask if
there exists an interaction between IL-22 stimulation of wild-type (WT) and Il22ra1-/- (KO) organoids,
by assuming the read count of sample i at gene j followed a negative binomial distribution:
Yij ~ NB(λijKi, θj),
with a mean parameter log(λij) = β0j + β1j(KO) + β2j(IL22) + β3j(KOxIL22),
where θj is the over-dispersion parameter and Ki is the size-factor for sample i obtained by Deseq
(Anders and Huber, 2010).
We performed a likelihood ratio test to assess the statistical significance of the KOxIL-22 interaction
term β3. Among the genes significantly up- or down-regulated by IL-22 (p-value < 10-12), we analysed
for enriched Gene Ontology terms within the category of biological processes using the Database for
Annotation, Visualisation and Integrated Discovery (DAVID) Functional Classification Tool
(http://david.abcc.ncifcrf.gov) (Huang da et al., 2009). Network analyses were subsequently
performed using GeneMania (Mostafavi et al., 2008) (integrating various interaction and network
databases including IntAct, BIND, MINT, BioGRID, REACTOME, etc…) and InnateDB (Lynn et al.,
2010).
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