Scale:chr2:
20 kb69,150,000 69,160,000 69,170,000 69,180,000 69,190,000 69,200,000 69,210,000
UCSC Genes (RefSeq, UniProt, CCDS, Rfam, tRNAs & Comparative Genomics)
GKN2 GKN1GKN3
Chromosome 2 (p13.3)21 p12 34 35
MouseRat
RabbitPig
DolphinCow
HorseCatDog
MicrobatHedgehog
ElephantArmadilloOpossum
Tasmanian_devilWallaby
Multiz Alignments (Mammalian Conservation)
GKN2
Figure S1. Mammalian conservation of GKN2. (A) UCSC genome browser output for the human GKN gene cluster (GKN1-3) with expanded view of the human GKN2 structural gene on chromosome 2p13.3 (GRCh37 Hg19; http://genome.ucsc.edu/). Mammalian conservation is shownin the ‘Multiz Alignment’ track. The degree of conservation, as shown in the grayscale density plot, uses blocks of darker values to indicate higher conservation.
Mouse models:
2m 12m
Autoimmune gastritis
Tumorigenesis (antrum)
Tumorigenesis (corpus)
H. pylori gastritis 7d
Atrophy/hypertrophy
HpSS1
GmcsfTg
gp130F/F
IL1βTg
HKβ-/-
Gastric epithelium
Mucus layer
Submucosa
Helicobacter pylori
NORMAL H. PYLORI GASTRITIS
GASTRICCANCER
Tumor
Inflammatory cells
INTESTINALMETAPLASIA
Goblet cells
Human GC progression:
Figure S2. Comparison of human and mouse GC progression. Schematic showing correspondence between stages of human GC as defined by Correa (Correa P et al. Lancet. 1975. 2(7924): 58-60) and five well characterised mouse models of gastric pathology: infection of C57BL/6 with mouse adapted H. pylori Sydney strain 1 (HpSS1); granulocyte-macrophage colony stimulating factor transgenic overexpression (GmcsfTg) model of autoimmune gastritis; H+K+ ATPase beta subunit knockout (HKβ-/-) model of corpus atrophy and hypertrophy; gp130Y757F cytokine co-receptor knock-in model of antral tumorigenesis (gp130F/F); interleukin-1 beta transgenic overexpression model of corpus tumorigenesis (IL1βTg).
A
274
124
151 Gkn2-/-Gkn2+/-WT
0
2
4
6
8
10
aver
age
litte
r siz
e
Birth Weaning
Gkn2-/-WT
B
Figure S3. Gkn2-/- mice show normal viability and fertility. (A) Pie chart shows genotype frequencies of progeny resulting from Gkn2+/- heterozygous pair matings. (B) Histogramshows mean litter sizes at birth and weaning from Gkn2-/- × Gkn2-/- matings compared to WT× WT matings. Error bars (SEM).
0
1
2
3
***
WTGkn2-/-
N
umbe
r of c
orpu
s le
sion
s
A
0
2
4
6
8
M
acro
scop
ic le
sion
are
a as
% o
f gas
tric
muc
osa
area ***
B
Figure S4. Quantitative morphometric analysis of corpus mucosal lesion number and macroscopic area. (A) Number of lesion foci per corpus stomach; (B) macroscopic lesion mucosa area as % of total gastric mucosal area in 12 week old Gkn2-/- (n=17) and WT littermate control (n=9) mice. Histograms show the mean. Error bars (+SEM). P values were determined using a 2-tailed Student’st test: ***(P<0.001).
-
Tff1 Gkn2 -/- /Tff1Gkn2 -/-WT -/- -/-A
0.0
0.1
0.2
0.3
0.4
0.0
0.1
0.2
0.3
0.4B
WTGkn2-/-
Tff1-/-
Gkn2-/-/Tff1-/-
Cor
pus
area
(mm
2 )/le
ngth
mus
cula
ris m
ucos
a (m
m)
Ant
ral a
rea
(mm
2 )/le
ngth
mus
cula
ris m
ucos
a (m
m)
ns
ns
C
Figure S5. Morphometric analysis of gastric epithelial lesions in Gkn2-/-/Tff1-/- mice.(A) Representative images of fundic and antral lesions in 12-week old Gkn2-/-/Tff1-/- compound mutant mice. Boundaries between fundic and antral mucosae are delineated with a white broken line; lesion areas are demarcated with either red (fundic) or green (antral) dotted outlines. Scale bar shows 5mm. (B-C) Histograms show the mean mucosal cross section (microscopic) area of fundic (B) and antral (C) mucosae. Error bars (+SEM). Abbreviations: ns; non-significant comparison in 2-tailed Student’s t test.
-
A
0
2
4
615202530
mRN
A fo
ld c
hang
e
Dmbt1 RegIIIγ
Gkn2-/- uninfected (4)WT H. pylori (13)Gkn2-/- H. pylori (18)
B
-5
0
5
10
15
D
IL10 Foxp3
mRN
A fo
ld c
hang
e
-20
0
20
40
60
80
100
mRN
A fo
ld c
hang
e
IL17f
C
mRN
A fo
ld c
hang
e (T
H1/
M1
gene
s)
-5
0
5
10
50
100
150
Ifnγ IL1β Cxcl2 IL6 IL11Tnfα
Figure S6. Lack of differential expression of cytokines and immune-related transcription factors in antral stomach of H. pylori-infected Gkn2-/- mice. QRT-PCR analysis of anti-microbial peptides Dmbt1 and RegIIIγ (A); Th-1/M1 cytokines (B), Th-17 cytokines (C) and Treg markers (D). Histograms show the mean mRNA fold change relative to WT uninfected control mice. Error bars + SEM.
C
A
Figure S7. Enhanced gastric immunity in Gkn2-/- mice is not cell autonomous to GECs. (A) Schematic showing the strategy used for co-culture of primary mouse GEC and H. pylori SS1. (B) Immunofluorescent co-localization of epithelial cells (pan-cytokeratins; Pan-CK), GKN2 and β-gal in cultured GECs prepared from Gkn2-/- and WT mice. Absence of contaminating immunocytes and mesenchymal cells was verified by CD45 and SMA staining respectively. Primary mouse embryonic fibroblasts (F’blasts) and peritoneal macrophage (Macs) cultures show corresponding positive controls for SMA and CD45. (C) Luminex bead array analysis of cytokines/chemokines in Gkn2-/- and WT GEC culture supernatants after co-culture with H. pylori for 24 hours. Histograms show mean levels (pg/mL). Error bars (+SEM). P values were determined using a 2-tailed Student’s t test. Statistical significance relative to WT uninfected controls: *P<0.05. There were no differences in chemokine/cytokine production between H. pylori-stimulated Gkn2-/- and WT GECs.
Gkn2-/-(n=9)
WT(n=7)
Primary GECs co-cultured with 1 x 10 cfu live H. pylori SS17
Gkn2-/- GECsWT GECs
0200400600800
100010002000300040005000
1000015000200002500030000
Cyt
okin
e le
vel p
g/m
L
WT uninfectedGkn2 -/- uninfectedWT H. pyloriGkn2-/-H. pylori
TNFαIL-
1αIL-
1β IL-5
IL-6
IL-10
IL-13
CCL3CCL4
CCL5
**
**
****
**
**
**
**
**
**
GK
N2
Pan
-CK WTWT WT
WT 50µm 20µm 50µmGkn2-/- 20µm
βgal
SM
AC
D45
GEC 50µm 20µm50µm
F’blasts
Macs
Nuc
Gkn2-/-
Pan
-CK
B
0
2
4
6
*
105
H. p
ylor
i CFU
/sto
mac
h
WT Gkn2-/-
Figure S8. Confirmatory analysis of H. pylori colonisation in Gkn2-/- mice by colony forming unit (CFU) assay. Histograms show mean H. pylori CFU counts per stomach at 7 days post infection. Error bars (+SEM). P values were determined using a 2-tailed Student’s t test: *(P<0.05). The CFU assay provides independent verification of QPCR data showing lower gastric colonisation levels in Gkn2-/- compared to WT mice.
-
0.00
0.02
0.04
0.06
0.08
** * * * *
GKN2 (DOX):
J99 ∆babA/sabA
- + - +H. pylori strain :
WT26 WT31 Empty Vector
J99 ∆babA/sabA
- + - +J99 ∆babA/
sabA
- + - +
H. p
ylor
i adh
esio
n/A4
50 n
m
A
B
50 100
200
500
10000
GKN2 (HA)
GAPDH
DOX (ng/mL)
DOX (ng/mL)
50 100
200
500
10000
WT26:
WT31:
GKN2 (HA)
GAPDH
nsns
ns
Figure S9. GKN2 is dispensable for H. pylori adhesion to GECs. (A) Stable transfected, GKN2-HA (C-terminal haemagglutinin epitope tagged) inducible MKN28 gastric epithelial cell lines were created using Tet-ON Advanced/TRE-tight expression vectors. Doxycycline (DOX) dependent induction of GKN2-HA expression was verified in two indepdent clones (WT26, WT31) by immunoblotting cell lysates with anti-HA antibodies. GAPDH blots confrim protein integrity. (B) ELISA-based detection of H. pylori J99 (WT strain) and ∆babA/∆sabA (adhesion-deficient isogenic mutant strain used as control) adherence to GKN2-HA inducible MKN28 gastric epithelial cells. Histograms show mean adhesion (absorbance units at 450nm (A450). Error bars (+SEM). P values were determined using a 2-tailed Student’s t test: *(P<0.05). H. pylori J99 adhesion was unaffected by GKN2 expression (non-significant; ns).
-
Figure S10. Antral cytokine protein expression in 7 day H. pylori-infected Gkn2-/- mice. Histograms show mean levels of pro- and anti-inflammatory cytokines measured by Luminex bead array in 7 day infected mice and uninfected controls (n=6 or 7/group). Error bars (+ SEM). P values were determined using a 2-tailed Student’s t test: *P<0.05.Abbreviations: nd; not detected.
050
100150200200300400500600
pg/1
00m
g pr
otei
n
TNFα IFNγIL-1α IL-6 IL-10CXCL1 CCL4
nd nd
WT uninfectedGkn2 -/- uninfected WT H. pyloriGkn2 -/- H. pylori
*
-
B
0
400
800
1200
E-Cad CD45
Cd4
5 m
RN
A fo
ld c
hang
e
0
20
40
60
E-Cad CD45Muc
5ac
mR
NA
fold
cha
ngeWT
Gkn2-/-
0
5
10
15
20
E-Cad CD45
Cdh
1 m
RN
A fo
ld c
hang
e
0
2000
4000
6000
8000
E-Cad CD45
IL6
mR
NA
fold
cha
nge
0
5000
10000
15000
20000
Ccl
4 m
RN
A fo
ld c
hang
e
0
5000
10000
15000
20000
25000
E-Cad CD45
IL1a
mR
NA
fold
cha
nge WT
Gkn2-/-
E-Cad CD45
C
0
1000
2000
3000
E-Cad CD45
Tnfa
mR
NA
fold
cha
nge
AC
D45
E-Cadherin
WT Gkn2-/-
CD45+ CD45+
E-Cad+ E-Cad+
Figure S11. Cell sorting and cytokine expression analysis of gastric epithelial and immune cells. (A) Representative FACS plots showing isolation of gastric epithelial (E-Cadherin+) and immune cell (CD45+) populations from WT and Gkn2-/- stomachs (n=3/group). (B) QRT-PCR validation of sorted cell populations for Cdh1 (E-Cadherin) and Cd45 mRNA expression. Analysis of Muc5ac mRNA confirms presence of SMC within the gastric epithelial population. (C) QRT-PCR analysis of pro-inflammatory cytokine expression in sorted gastric epithelial and immune cell populations. IL1a, Tnfa, IL6 and Ccl4 mRNA showed specific enrichment in gastric immune cellsand were undetected in the epithelial cells. Error bars (+SEM)._
Ly6C
CD11b+ CD49d+ CD11b+ Ly6CHi
Ly6G- CD49d+
Mo-MDSC
Ly6G
CD49d
Ly6G
FSC
SS
C Leukocytes
CD
11b
F4/80
CD11b+
CD
11b
CD
11b
F4/80Ly6C
CD11b+ CD11c+
DCMacrophages
CD11b+ F4/80+
Gr1-Ly6G
FSC
SS
C
CD11c
LeukocytesGr1-
Gr1-
B
A
Figure S12. Flow cytometric gating strategy to resolve gastric mucosal myeloid cell populations. Initial gating on viable cells by propidium iodide dye exclusion (not shown). Subsequent gating was on total gastric leukocytes (forward/side scatter) for (A) macrophages, dendritic cells (DC) and (B) monocytic myeloid derived suppressor cells (Mo-MDSC) are shown.
-
WT uninfected Gkn2 -/- uninfected WT H. pylori Gkn2 -/- H. pylori
CD11b+ Ly6CHi Ly6G- CD49d+
10.9
Ly6G
Ly6C
2.7 8.7 2.8
Mo-MDSC
CD
11b
F4/80
4.6 12.1 3.9 9.5
CD11b+ F4/80+ Gr1-Macs
CD
11b
CD11c
CD11b+ CD11c+
3.4 7.0 3.2 5.7DC
Figure S13. Flow cytometric analysis of gastric mucosal immune cells in 7 day H. pylori infected Gkn2-/- mice. Representative flow plots showing key myeloid cell subsets in stomachs of 7 day infected Gkn2-/- mice and uninfected controls (n=5/group): macrophages (Macs; CD11b+F4-80+Gr1-),dendritic cells (DC; CD11c+), monocytic (Mo)-MDSC (CD11b+Ly6ChiLy6G-CD49d+).
WT Gkn2+/- Gkn2-/-A
Figure S14. Baseline gastric phenotype of Gkn2+/- mice. (A) Macroscopic images of stomachs from 12 week old mice. White broken lines delineate boundaries of corpus and antral mucosae. Scale bar 5 mm. (B) Histology of AB-PAS stained corpus and antrum mucosa in 12 week old mice. Scale bars 50 µm. (C) Morphometric analysis of corpus and antral mucosal thickness in 12 week old WT (n=8); Gkn2+/- (n=4); Gkn2-/- (n=11) mice. (D) Ki-67 labelling of proliferating cells in corpus mucosa of 12 week old WT (n=6); Gkn2+/- (n=4) and Gkn2-/- (n=6) mice. Error bars (+SEM). P values weredetermined using a 2-tailed Student’s t test: *(P<0.05). Gkn2+/- mice show a normal gastric phenotype in macroscopic appearance, mucosal histology/thickness and epithelial proliferation.
-
0.0
0.1
0.2
0.3
0.0
0.1
0.2
0.3
0.4
0.5
Muc
osal
are
a (m
m2 )/
leng
thm
uscu
laris
muc
osa
(mm
)
WT
Gkn2-/-Gkn2+/-
Corpus AntrumC
0
10
20
30
% K
i-67
posi
tive
cells
/gla
nd
WT
Gkn2 -/-Gkn2+/-
CorpusD
**
B
Cor
pus
Ant
rum
WT Gkn2+/- Gkn2-/-
A
Gkn2-/-(n=18)
WT(n=13)
(6 - 8 weeks old)
Oral delivery of 1 x 10 cfu H. pylori SS1
2
Sacrifice
0
7
Infection timecourse/months
Gkn2+/-(n=24)
Gkn2 H. pylori+/-WT H. pylori Gkn2 H. pylori-/-C
0.0
0.5
1.0
1.5
2.0
2.5
PMN MN Atrophy PMN MN
Corpus Antrum
MetPa
thol
ogy
Scor
e
DGkn2 -/- uninfected WT H. pylori
Gkn2 -/- H. pyloriGkn2+/- H. pylori
#
#
##
##
#
# # #
#
#
** * *
**
#
##
Figure S15. Gastric immunopathology in H. pylori infected Gkn2+/- mice. (A) Schematic showing strategy used for 2 month H. pylori infections and treatment group sizes. (B) QPCR analysis of H. pylori SS1 colonisation levels in 2 month infected gastric homogenates. (C) Macroscopic and corresponding histological views gastric corpus from 2 month H. pylori infected mice. Scale bars: 5 mm (macro); 50 µm (histology). (D) Summary of semi-quantitative gastric histopathology. (E) QRT-PCR analysis of TH1/proinflammatory (Ifng, IL1b, IL6, Cxcl2), anti-inflammatory (IL10), TH17 (IL17f) cytokine mRNA expression in 2 month infected gastric corpus. Error bars (+SEM). P values were determined using a 2-tailed Mann Whitney U test (Figure S12B) or a 2-tailed Student’s t test (Figure S12D-E). Statistical significance compared to WT uninfected control mice: #(P<0.05). Statistical significance between treatment groups: *(P<0.05); **(P<0.01).
-
0
0.5
1.0
1.5
104
H. p
ylor
i gen
omes
/10
5 G
apdh
cop
ies
B
*
* WT H. pylori
Gkn2 -/- H. pyloriGkn2+/- H. pylori
-5
0
5
10
15
20
25
30
Ifng
mR
NA
fold
cha
nge
-2
0
2
4
6
8
IL1b
mR
NA
fold
cha
nge
0
40
80
120
IL6
mRN
A fo
ld c
hang
e
0
2
4
6
8
IL10
mRN
A fo
ld c
hang
e
0
10
20
30
40
Cxc
l2 m
RNA
fold
cha
nge
-50
0
50
100
150
200
250
IL17
f mRN
A fo
ld c
hang
e
E#
#
#
***
# #
#*
#
##
#
##
#
###
#
##
* ***
Gkn2 -/- uninfected WT H. pylori
Gkn2 -/- H. pyloriGkn2+/- H. pylori
Table S2. Gkn2-/- mice show normal fertility. Shown are average litter sizes at
birth and weaning produced by Gkn2-/- × Gkn2-/- matings compared to WT × WT
matings. P value (Pval) significance levels from unpaired t-tests are shown. Litter
sizes resulting from Gkn2-/- matings did not differ significantly from those resulting
from WT matings.
Parents
genotype
Litters (n) Av. litter size
(at birth)
Pval Av. litter size
(at weaning)
Pval
WT 28 7.39 0.408 4.64 0.820
Gkn2-/- 44 6.86 4.44
Table S1. Gkn2 null alleles show normal Mendelian inheritance. Shown
are observed versus expected genotype frequencies for progeny produced by
matings between Gkn2+/- heterozygous pairs, chi-squared (2) statistic and P
value (Pval) significance level. Observed genotypes did not deviate
significantly from expected Mendelian ratios.
Genotype Observed
(((Expected)
% of Total 2 (deg. freedom) Pval
WT 151 (137) 27.5 2.668 (2) 0.26
Gkn2+/- 274 (275) 49.9
Gkn2-/- 124 (137) 22.6
Supplemental Methods
Loss of Gastrokine-2 drives premalignant gastric inflammation and
tumor progression
Trevelyan R. Menheniott, Louise O’Connor, Yok Teng Chionh, Jan Däbritz,
Michelle Scurr, Benjamin N. Rollo, Garrett Z. Ng, Shelley Jacobs, Angelique
Catubig, Bayzar Kurklu, Stephen Mercer, Toshinari Minamoto, David E. Ong,
Richard L. Ferrero, James G. Fox, Timothy C. Wang, Philip Sutton, Louise M.
Judd, Andrew S. Giraud
Taqman Q-PCR based determination of H. pylori colonisation level
Genomic (g)DNA was extracted from H. pylori-infected mouse stomach tissue using Trizol reagent (Invitrogen) and diluted 1:10 in 10 mM Tris-HCl buffer. Diluted gDNA was analysed by Q-PCR quantitation of H. pylori SS1 genome copies (16S rRNA gene) relative to mouse Gapdh copies as described (1) using a ABI 7500 Fast Real Time PCR analyser (fast cycling) and ABI Taqman fast reagents (Applied Biosystems) in 12 µL reactions. Cycling parameters and sequences of oligonucleotide primer pairs/fluorogenic hybridisation probes were as follows:
Initial denaturation: 95˚C for 10 mins 40 cycles: 95˚C for 3 secs 60˚C for 30 secs (fluorescence data capture)
ELISA-based detection of anti-H. pylori antibodies in mouse serum
Levels of circulating anti-H. pylori antibodies were quantified by standard direct enzyme-linked immunosorbent assay (ELISA) as described (2). Briefly, Maxisorp Immunoplates (Nunc, Roskilde, Denmark) were coated overnight with 10 μg/well of H. pylori SS1 lysate in 100 μL of bicarbonate buffer (pH 9.6). After being washed three times with tap water then one time with 0.05% (v/v) Tween 20 in PBS, wells were blocked with 1% (w/v) bovine serum albumin in PBS (PBS-BSA) for 1 hr at room temperature. Blocker was washed off as above then mouse serum samples were serially diluted 1:100 to 1:108 in PBS-BSA and 100 μL added to duplicate wells before incubation at room temperature for 30 min. After further washing, 100
Primer name Sequence (5’ fluorochrome and 3’ quencher dyes)
16S rRNA-F TTTGTTAGAGAAGATAATGACGGTATCTAA C
16S rRNA-R CATAGGATTTCACACCTGACTGACTATC
16S rRNA-Probe FAM-5'-CGTGCCAGCAGCCGCGGT-3'-TAMRA
Gapdh-F TGCACCACCAACTGCTTAG
Gapdh-R GGATGCAGGGATGATGTTC
Gapdh-Probe FAM-5'-CAGAAGACTGTGGATGGCCCT-3'-TAMRA
2
μL of either horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G1 (IgG1) diluted 1/6,000 (Southern Biotech, Birmingham, AL, USA) or HRP-conjugated goat anti-mouse IgG2c (Immunology Consultants Laboratory, Portland, OR, USA) diluted 1/5,000 in PBS-BSA was added to each well and the plates incubated at room temperature for 1 hr. Following additional washes color was developed by addition of 100 μL tetramethylbenzidine (Zymed, CA) and the reaction stopped by addition of 100 μL of 2M H2SO4. Absorbance was read at 450 nm and end point titers calculated. The observed end point titer is the highest serum dilution that yielded an optical density (OD) greater than the value that defined the cutoff between positive and negative results. The cutoff value is determined by calculating the mean of the OD of the negative control wells (PBS-BSA only) for each plate + 2 standard deviations (IgG1) or 3 standard deviations (IgG2c). Titer is expressed as the reciprocal dilution of the OD cutoff value for each sample. Where the observed values failed to reach the OD of the lowest dilution (1:100) the titer was assigned the minimum dilution value of 100.
ELISA-based determination of H. pylori adhesion to cultured human GECs
MKN28 gastric epithelial cells were cultured in 96-well plates at 37°C and 5% CO2 for 48 hrs. Growth medium was aspirated and the cells co-incubated with 107 cfu
H. pylori J99 (3) or adhesion defective H. pylori J99 babA/sabA isogenic mutant (gifts of Michael McGuckin, Mater Medical Research Institute, Brisbane, Australia; ref. (4)) in Dulbecco’s modified Eagle medium (DMEM) and the plate gently agitated for 30 min at 37°C. Cells were fixed with 1% paraformaldehyde in PBS for 10 min. Positive controls (maximal H. pylori binding to wells) comprised wells with no gastric cells, to which bacteria were added and allowed to adhere to the plastic before fixation. Negative controls contained neither gastric cells nor bacteria. After washing, cells were blocked for 30 min with PBS-BSA and bacterial adhesion determined by ELISA using hyperimmune mouse serum (generated in house) and a goat anti-mouse IgG-HRP secondary antibody (Pierce; 1/10,000) for detection. Binding was visualized by addition of 100 μL tetramethylbenzidine (TMB) substrate per well, with absorbance read at 450 nm. Generation of polyclonal antiserum Antibodies were prepared essentially as described (5). To generate anti-mouse GKN2 antibodies, a custom immunizing peptide 12-mer corresponding to the C-terminus of mouse GKN2 was synthesized (sequence: ILGVSICGGIH; molecular weight 1068 Daltons; Auspep, Australia) at >95% purity. To generate anti-human GKN2 antibodies a custom 10-mer corresponding to the C-terminus of human GKN2 was synthesized (sequence: GISICADIHV; molecular weight 1030 daltons; Auspep, Australia) at >95% purity. Mouse and human GKN2 immunising peptides were respectively coupled to key-hole limpet haemocyanin (KLH; Sigma) with 5% glutaraldehyde using 1 mg immunising peptide, 2 mg KLH and 80 µL glutaraldehyde in 1 mL PBS for 1 hr. The conjugate was dialysed overnight against PBS using 6000MW pore dialysis tubing to remove free peptide and glutaraldehyde, then frozen in aliquots for immunization. New Zealand white rabbits (two pairs; Walter and Eliza Hall Institute (WEHI) Antibody Facility, Bundoora, Victoria, Australia) were bled from a marginal ear vein (pre-immunization antibody titer) then immunized intramuscularly with 100 nM human or mouse GKN2-KLH conjugate emulsified in two volumes of complete Freund’s
3
adjuvant (Sigma). Five weeks later rabbits were boosted with 50 nM GKN2-KLH conjugate in incomplete Freund’s adjuvant (Sigma) and bled 7-10 days later (5-20 mL volumes). Two subsequent immunizations and bleeds were carried out at intervals depending on antibody titer using incomplete Freund’s adjuvant. The final bleeds (anti-mouse GKN2-R771 B3; anti-human GKN2-R779 B3) was used in all subsequent experiments at a final dilution of 1:500 (immunohistochemistry and immunofluorescence cytochemistry) and 1:3000 (immunoblotting). Co-culture of primary gastric epithelial cells with H. pylori
Primary mouse gastric epithelial cell (GEC) cultures were derived as described (6). Briefly, stomachs from 3 week old mice were dissected, opened and contents rinsed away with sterile PBS. Stomachs were transferred to 0.2% w/v bovine serum albumin (BSA) in Hanks buffered salts solution (HBSS; Sigma), chopped into ~1 mm3 pieces then digested by incubation in 0.4 mg/mL collagenase A (Roche) in HBSS at 37°C for 1 hr. Digested tissue was resuspended in DMEM Glutamax medium supplemented with 20% FBS, 2 mM non-essential amino acids, 50 IU penicillin, 50 ug/mL streptomycin (Invitrogen), disaggregated by repeated pipetting and seeded into 24-well plates. After one hr at 37°C with 5% CO2/air to allow for the attachment of contaminating fibroblasts, the unattached fraction (enriched for GECs) was aspirated, seeded in fresh 24-well plates and incubated for a further 48 hrs to allow for GEC attachment. Cells were co-cultured with 1 × 107 cfu live H. pylori SS1 for 24 hrs as described (7) after which culture supernatants were harvested for cytokine analysis. Genotyping assay for the Gkn2-(lacZ) knock-in allele
Mice were genotyped by end point PCR from ear punch biopsies using the following cycling parameters and multiplexed oligonucleotide primer sequences: Initial denaturation:
95˚C for 3 mins 35 cycles: 95˚C for 30 secs 60˚C for 1 min
72C for 1 min Final extension:
72C for 5 min
Products were analysed by electrophoresis through 1.5% agarose, Tris-borate EDTA (TBE) gels. Gkn2-(WT)-F1 and Gkn2-(WT)-R1 primers amplify a 442 bp product from the Gkn2 WT allele, Gkn2-(WT)-F1 and Gkn2-(lacZ)-R2 primers amplify a 214 bp product from the Gkn2-lacZ knock-in allele. WT, Gkn2-/- and
Primer name Sequence
Gkn2-(WT)-F1 CAGTCCTGATAAGGGAGTTTGC
Gkn2-(WT)-R1 CCCTCACAAAGTGATGAGAAGG
Gkn2-(lacZ)-R2 ATTCTCCCAATCTCTCCTCTGC
4
Gkn2+/- mice were respectively discriminated by amplification of either 442 bp, 214 bp or both 442/214 bp products (see Figure 2B, main text).
Generation of inducible human GKN2 expressing gastric epithelial cell lines
To generate MKN28 gastric epithelial cell lines with an inducible GKN2 protein expression cassette we used the Tet-ON Advanced/TRE-tight binary vector system (Clontech). A full length human GKN2 cDNA was amplified from human gastric antrum cDNA with simultaneous engineering of a C-terminal haemagglutinin (HA) epitope tag and flanking NotI-SalI restriction sites by PCR mutagenesis. Oligonucleotide primer sequences are shown in the table below with color coding used to indicate HA-tag sequence, NotI and SalI restriction sites; stop codons. Kozak consensus (bold type); GKN2 coding sequence (underlined).
Primer name Sequence
NotI-GKN2-F 5’-GCGGCCGCACCATGAAAATACTTGTGGCATTTCTGG-3’
SalI-stop/stop-HA-GKN2-R 5’-GTCGACTCACTAAGCGTAATCTGGAACATCGTATGGG- -TAAACATGAATGTCTGCACAGATTGAAATTCC-3’
Initial denaturation:
95C for 3 mins 30 cycles:
95C for 30 secs
55C for 1 min
72C for 1 min Final extension:
72C for 5 min PCR-engineered GKN2-HA cDNA product (750 bp) was purified by electrophoresis through a 1% agarose, Tris-acetate EDTA (TAE) gel, subcloned into the pGEM-T-easy vector and correct mutagenesis verified by sequencing using BigDye_v3.1 chemistry (Applied Biosystems). The GKN2-HA cDNA was excised by double digestion with NotI and SalI then directionally ligated into the NotI-SalI sites of the pTRE-tight expression vector. MKN28 cells were maintained in RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS), 50 IU/mL penicillin, 50 g/mL streptomycin (Invitrogen) at
37C with 5% v/v CO2/air (5). MKN28 cells were transfected with 2 g linearised pTet-ON Advanced vector DNA using FuGENE HD (Roche), incubated for 48 h,
then subcultured (1:10 split) and stable transgenic clones selected with 300g mL-1 G418 (Sigma) for two weeks. G418-resistant colonies were trypsinised and cloned by single cell deposition into 96-well plates using a Mo-Flo sorter (Beckman-Coulter). MKN28 Tet-ON clones with inducible potential were identified by supertransfection with pTRE-tight-luciferase in the presence and absence of 1 µg/mL doxycycline (DOX) inducer. Two clones (F6 and G6) showing robust DOX-dependent luciferase induction/repression were expanded into stable Tet-ON cell
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lines. MKN28 Tet-ON F6/G6 cells were transfected 2 g linearised pTRE-tight-GKN2-HA as described above and double stable transgenic clones selected with
150 g/mL G418 (maintenance dose) and 200 g/L hygromycin for two weeks. G418- hygromycin double resistant colonies were single cell cloned as described above and expanded into MKN28 Tet-ON/TRE-tight-GKN2-HA continuous cell lines. Two clones (WT26 and WT31) showing appropriate DOX-dependent induction of GKN2-HA expression were used in all experiments.
Gene expression (QRT-PCR) cycling parameters and primer sequences
QRT-PCR was performed using an ABI 7500 Fast Real Time PCR analyser (Applied Biosystems) in standard cycling mode and Go-Taq SYBR-green reagents (Promega) in 20 µL reactions. Cycling parameters and oligonucleotide primer sequences used for mouse and human gene expression analysis were as follows:
Initial denaturation:
50˚C for 2 mins 95˚C for 10 mins 40 cycles: 95˚C for 15 secs 60˚C for 1 min (fluorescence data capture) Dissociation curve analysis:
60˚C 95˚C 1% increment/30secs
Human primer sequences:
Mouse primer sequences:
Gene Forward Primer Sequence Reverse Primer Sequence
Rpl32 GAGGTGCTGCTGATGTGC GGCGTTGGGATTGGTGACT
Gkn2 GAGACAGTGACCATCGACAACC GAGACAGTGACCATCGACAACC
Muc6 GCTGTGTATGACAAGTCGGGTTAC AATTTTGTCCTTCTTGGACAGATACAT
Mist1 CCAGGGTGCTCCTTCTTTTG GGCGGAAGTTCACCATCCTT
HKβ CCGGTGGGTGTGGATCAG GCAAAGAGCCCGGTCATG
Ifn CCAGGACCCATATGTAAAAGAAGC TCATGTCTTCCTTGATGGTCTCC
IL1β ATGACCTGAGCACCTTCTTTCC CTTGTTGCTCCATATCCTGTCC
Tnfα GTAGCCCACGTCGTAGCAAA ACAAGGTACAACCCATCGGC
Cxcl2 AGTGAACTGCGCTGTCAATG TTCAGGGTCAAGGCAAACTT
IL6 ACAAAGCCAGAGTCCTTCAGAGA CTGTTAGGAGAGCATTGGAAATTG
Gene Forward Primer Sequence Reverse Primer Sequence
GAPDH GACATCAAGAAGGTGGTGAAGC GTCCAACCCTGTTGCTGTAG
GKN1 GGCCTGATGTACTCAGTCAACC TTTAGTTCTCCACCGTGTCTCC
GKN2 TGCAGGATCATGCTCTTCTAC TGGTCCATCTTCAGGATAAAG
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IL11 TCATTCAGGGAGGCTAAGGA ACAGGGTTTTGCCATGTCTC
Dmbt1 GCTCCACAAGCACAGATTCC TGCCATCTCATTGTTCTTGG
RegIII ACGAATCCTTCCTCTTCCTCAG GTCTTCACATTTGGGATCTTGC
IL10 TGATGCCCCAGGCAGAGA CACCCAGGGAATTCAAATGC
Foxp3 TTCACCTATGCCACCCTT ATCC TTTCTGAAGTAGGCGAACATGC
IL4 GTCATCCTGCTCTTCTTTCTCG TCACTCTCTGTGGTTCTTCG
IL5 TTGACAAGCAATGAGACGATGAG GCCCCTGAAAGATTTCTCCAA
Fizz1 CCTGCTGGGATGACTGCTACT CTCCACTCTGGATCTCCCAAGAT
Ym1 TCTGGTGAAGGAAATGCGTAAA GCAGCCTTGGAATGTCTTTCTC
IL1RN CATAGTGTGTTCTTGGGCATCC TCAGAGCGGATGAAGGTAAAGC
IL17a TCTGTGTCTCTGATGCTGTTGC ACATTCTGGAGGAAGTCCTTGG
IL17f AAAACCAGGGCATTTCTGTCC ACCAGGATTTCTTGCTGAATGG
Ccr2 AAGTTCAGCTGCCTGCAAAGAC GCCGTGGATGAACTGAGGTAAC
IL4ra GTGCCACATGGAAATGAATAGG TGTGAGGTTGTCTGGAGCTAGG
S100a8 GTCCTCAGTTTGTGCAGAATATAAA GCCAGAAGCTCTGCTACTCC
S100a9 CTCTTTAGCCTTGAAGAGCAAG TTCTTGCTCAGGGTGTCAGG
Arg1 AAAGCTGGTCTGTGGAAAA ACAGACCGTGGGTTCTTCAC
Arg2 ACCCTTGCGGCTCACACA AGCAGGATTGCAGACACTGAAA
Tgfb1 GGTGTGGTCTATGCCATCAA CCTCTCCAGTAACCGCTGAT
Tgfb2 CTTCACCACAAAGACAGGAACC CCATCAATACCTGCAAATCTCG
RoRt GAGTTTGCCAAGCGGCTTT TCCATTGCTCCTGCTTTCAGT
Tbet TCCAAGAGACCCAGTTCATTGC CGTATCAACAGATGCGTACATGG
Tff1 AGAGGTTGCTGTTTTGATG AGTCTGAGGGGTTGAACTG
Tff2 CCCCACAACAGAAAGAAC GGGCACTTCAAAGATCAG
Muc1 CACACTCACGGACGCTACGT TACCTGCCGAAACCTCCTCAT
Muc5ac GCAGTTGTGTCACCATCATCTGTG GGGGCAGTCTTGACTAACCCTCTT
Quantitative morphometry
Quantitative morphometry was performed by a blinded observer in all cases. At least six representative photographs per animal (n≥5) of standard histological or immunohistochemistry sections were captured using a Nikon Eclipse 80i light microscope equipped with a DS Ri1 imaging system (Nikon). To generate morphometric measurements, lengths, areas or relevant cells were manually outlined within images using ImageJ software for Windows v1.44p (http://rsb.info.nih.gov/ij/index.html). All measurements were converted from image pixels to mm (lengths) or mm2 (areas) by comparison with a calibrated graticule.
Determination of gastric acid content
For determination of gastric acid content in mice, stomachs were dissected and clamped at the oesophagus and duodenum prevent leakage of contents. Stomachs were injected with 2mL normal saline (0.9% w/v NaCl), incubated at room temperature for 5 mins with agitation, then cut open and the contents collected in
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sterile tubes. The pH of the stomach contents was determined immediately using a pre-calibrated pH meter (3510, Jenway).
Definitions of gastric histopathology
Corpus and antral inflammation was defined by sub-mucosal and mucosal presence of polymorphonuclear and mononuclear cells. Defects in gastric epithelial differentiation were assessed by several criteria. Corpus glandular atrophy was defined as loss of parietal and zymogenic cells. Mucus neck cell (MNC) hyperplasia was defined as expansion of the neutral mucin-secreting MNC zone, without ectopic presence of acidic mucins. MNC hyperplasia is a low grade lesion that may precedes the emergence of mucus metaplasia (see below). Surface mucus cell (SMC) metaplasia describes a lineage having the morphological characteristics of SMC, but showing loss of typical neutral mucins and ectopic gain of Alcian blue-stained acidic mucins. This atypical metaplastic phenotype was frequently found in Gkn2-/- mice, particularly in association with MNC hyperplasia, but has not been widely described elsewhere. Mucus metaplasia was defined as the ectopic presence of Alcian blue stained acidic mucins associated with the acquisition of an elongated antral glandular structure within corpus glands. This metaplasia is a premalignant change, commonly observed in chronic H. pylori infections of humans and mice, as well as some mouse models of GC.
Scoring criteria for semi-quantitative gastric histopathology
Inflammation (corpus and antrum) was scored according to the extent of immune cell infiltration within the fundic mucosa: 0, no immune cells were detected in the submucosa and mucosa; 0.5, few immune cells located within the submucosa only; 1, immune infiltration of the submucosa with or without infiltration at the very base of the mucosa; 2, immune infiltration of the submucosa and the bottom half of the mucosa; 3, immune infiltration of the submucosa and greater than 50% of the mucosa; 4, transmural infiltration of immune cells. Glandular atrophy was scored based on the estimated percentage of parietal cell loss and chief cell loss within the corpus: 0, no visible parietal cell and chief cell loss; 1, 25% parietal cell loss and 50% chief cell loss; 2, 50% parietal cell loss and greater than 75% chief cell loss; 3, 75% parietal cell loss and 100% chief cell loss; 4, greater than 75% parietal cell loss and 100% chief cell loss. Mucus neck cell (MNC) hyperplasia was scored according to the approximate percentage of corpus mucosa encompassed by glandular units with an expanded MNC zone: 0, 0% of mucosa affected; 1, 25% of mucosa affected; 2, 50% of mucosa affected; 3, 75% of mucosa affected; 4, 100% of mucosa affected. Surface mucus cell (SMC) metaplasia severity was scored according to the proportion of surface mucus cells affected: 0, no visible surface mucus cell metaplasia; 1, small foci were affected; 2, up to one-third of SMC were affected; 3, one-third to two-thirds of SMC were affected; 4, greater than two-thirds of SMC were affected. Mucus metaplasia (H. pylori infected mice only) was scored based on the approximate percentage of the corpus mucosa showing replacement of oxyntic glands with elongated Alcian-blue stained glands reminiscent of antral mucosa: 0, no visible mucus metaplasia; 1, small foci were present; 2, up to one-third of the corpus was affected; 3, one to two thirds of the corpus were affected; 3, greater than two thirds of the corpus were affected.
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Immunoblotting and multiplex cytokine array analysis
Tissue protein extracts, cell lysates and culture supernatants were size fractionated by 15% SDS-PAGE, transferred to nitrocellulose membranes (GE Healthcare) and blocked in 5% non-fat milk powder/Tris-buffered saline (TBS) pH 7.4, 0.1% Tween-
20. Membranes were incubated with primary antibodies overnight at 4C. Detection was performed with anti-rabbit/ anti-mouse IgG-HRP conjugates (DAKO) and enhanced chemiluminescence reagents (GE Healthcare). Sources of primary antibodies: rabbit polyclonal anti-mouse GKN2 (R771-B3) diluted 1:3000; and anti-human GKN2 (R779-B3) diluted 1:3000 (custom generated, see above); rabbit polyclonal anti-human GAPDH (Abcam, #ab9485) diluted 1:3000. Cytokine protein content of mouse stomach tissues and cell culture supernatants was determined using Bio-Plex Pro Mouse 23-plex cytokine bead arrays (Bio-Rad) according to the manufacturer’s protocols. In brief, stomach tissues were homogenized in ice cold BHI supplemented with complete mini protease inhibitor cocktail (Roche), homogenates cleared by centrifugation, supernatants collected and diluted 1:4 in BHI prior to analysis. Cell culture supernatants were centrifuged to remove cell debris. A nine point standard curve was generated by serial dilution of normalized cytokine standard in diluents specific to each assay (i.e. BHI broth or RPMI/DMEM culture medium) to ensure that the matrix used for assay calibration matched that of the samples. Samples were added in 50µL volumes either diluted 1:4 (stomach homogenates) or undiluted (culture supernatants). Plates were scanned on a Luminex 200 analyzer using xPONENT software (Luminex Corporation). Whole mount staining for β-galactosidase in situ
Stomach tissue expression of the lacZ reporter gene was assessed by histochemical staining for encoded β-galactosidase activity as described (8). Stained tissues were post fixed in 4% paraformaldehyde (Sigma) in PBS overnight at room temperature and then processed for standard paraffin histology as described (9). Supplemental Methods References
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