Supplemental methods
Detection and quantification of apoptotic cells
To detect apoptotic cells in tissues, labeling of degraded DNA in apoptotic cells was
performed using a modification of the terminal deoxynucleotidyl transferase-mediated
deoxyuridine triphosphate nick end-labeling (TUNEL) technique and in-situ cell
death detection kits (Roche Diagnostics), according to the manufacturer’s instructions.
Apoptosis were quantified by counting the number of TUNEL-positive cells per 100
nuclei and the apoptotic index was calculated as the ratio of apoptotic to total cells.
Mouse whole colon cultures
Colon tissue (200-300 mg) was washed in cold PBS containing penicillin and
streptomycin and cut into small pieces (0.5×0.5 cm), which were cultured (three
pieces per mouse) in 24-well flat bottom culture plates in serum-free RPMI 1640
medium (Gibco) at 37ºC for 24 h. The culture supernatants were then centrifuged at
9000 g at 4ºC for 5 min and stored at -80℃ until use.
Histopathology and immunohistochemistry
The colon was removed, fixed in 10% neutral-buffered formalin solution, embedded
in paraffin, and cut into tissue sections, which were stained with hematoxylin and
eosin. For immunohistochemistry, paraffin-embedded slides were deparaffinized,
immersed overnight in a 80ºC water bath in 10 mM sodium citrate buffer containing
0.1% Tween 20 for antigen unmasking, and incubated for 2 h at 37ºC in blocking
buffer A [phosphate-buffered saline (PBS) containing 1% BSA and 10% goat serum
(Serotech)]. They were then incubated for 2 h at 37ºC with primary antibody against
human IL-6 (Abcam), IL-1β (Abcam), HNP1-3 (Hycult Biotech.), CD68 (Dako), Gr-1
(BD Biosciences) or Ki-67 (Dako) in blocking buffer A, then for 1 h at room
temperature with biotinylated secondary antibody (Dako) in blocking buffer A.
Streptavidin-HRP (BD Pharmingen) in PBS was then added for 40 min at 37ºC and
the sections stained with DAB substrate and counterstained with hematoxylin.
Positive cells were counted in 5 randomly selected visual fields per section of each
samples at 400× magnification.
Immunoblotting
Samples were homogenized and sonicated in RIPA lysis buffer (Santa Cruz Biotech.),
supplemented with protease inhibitors. After centrifugation at 20,000 g for 15 min at
4ºC, protein in the supernatant was measured using a BCA protein assay kit (Pierce)
and 30 μg of protein were dissolved in reducing Laemmli sample buffer and separated
on 10% SDS-polyacrylamide gels and transferred onto an Immunobilon-P Transfer
membrane (Millipore). After blocking for 1 h at room temperature in blocking buffer
B (5% skim milk in Tris-buffered saline containing 0.05% Tween 20), the membrane
was incubated for 2 h at room temperature with primary antibodies at a 1:1000
dilution in blocking buffer B, then for 1 h at room temperature with immunoPure
peroxidase-conjugated anti-rabbit IgG antibodies (DAKO) in blocking buffer B. The
membrane was then treated with Super Signal West Dura Extended Duration
Substrate (Pierce) and signals detected using a LAS-3000 mini CCD camera (Fuji
Film). All primary antibodies used for immunoblotting were purchased from Cell
Signaling, except the anti-murine IL-1β antibody (Biolegend).
Isolation of mouse and human colonic epithelial cells
The entire length of the colon was opened longitudinally, washed with PBS
containing 100 μg/ml of gentamycin, and immersed in fresh ice-cold PBS containing
protease inhibitors and phosphatase inhibitors (0.2 mM phenylmethylsulfonyl fluoride,
5 μg/ml of aprotinin, 1 mM benzamidine, 1 mM sodium orthovanadate, and 2 μM
cantharidin). All subsequent steps were carried out at 4ºC. The colon was cut into
small pieces, which were immersed in 5 ml of ice-cold BSS buffer (1.5 mM KCl, 96
mM NaCl, 27 mM sodium citrate, 8 mM KH2PO4, 5.6 mM Na2HPO4, 15 mM EDTA,
and 1 mM dithiothreitol, containing the same inhibitors as above) and vortexed at
maximum speed for 3 x 20 min, then the material was passed through a 200 m mesh
nylon filter and the filtrate, containing epithelial cells, removed and retained. The
material retained on the mesh was then suspended in 5 ml of BSS buffer and vortexed
and filtered again as described above and the filtrate retained. This process was
repeated once more, yielding material still retained on the filter (used for the isolation
of lamina propria cells described below) and 3 suspensions of epithelial cells, which
were pooled and centrifuged at for 10 min at 800 g, and the pellet washed once with
150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM Tris-HCl, pH 8.0, containing the
above inhibitors. The final pellet was then collected as colonic epithelial cells [purity
＞80%, shown by staining with antibodies against the epithelial cell marker CK-18
(eBiosciences)], which were stored at -80 °C for protein and gene expression
experiments.
Isolation of mouse spleen cells, mouse and human lamina propria immune cells,
and human blood neutrophils and in vitro treatment
A splenic single cell suspension was obtained by crushing the spleens of control and
CAC mice through a 200 m mesh nylon filter. To isolate human and mouse LP
immune cells, the material left on the nylon mesh after the preparation of colon
epithelial cells was washed with Hank’s balanced salt solution (Gibco) and digested
by shaking at 37°C for 1 h with RPMI 1640 medium containing 100 U/ml of
collagenase D (Roche Diagnostics). The digestion was repeated 2 more times, then
the pooled digest supernatants were centrifuged at 800 g for 10 min at 4°C and
washed with PBS. The pellet was then suspended in 40% Percoll and layered on a
40%/100% step Percoll gradient (Pharmacia) and spun at 1,000 g for 5 min to collect
the lymphocyte-enriched population at the 40%/100% Percoll interface as LP immune
cells. LP mononuclear phagocytes (MPs) were then isolated by sorting for Gr-1lo/CD11b+ cells and neutrophils were isolated by sorting for Gr-1hi CD11b+ cells.
Neutrophils were also prepared from the peripheral blood of healthy human
volunteers by sorting for Gr-1hi CD11b+ cells.
FACS-sorted neutrophils (5×104/well) were seeded in 24-well flat-bottomed culture
plates and incubated for 24 h with 100 ng/ml of recombinant LPS (Sigma-Aldrich). In
some cases, neutrophils were cocultured for 24 h with LP MPs from normal mucosa
at a ratio of 1:2. To assess a direct role of IL-1 signaling in driving IL-6 expression
by MPs, IL-1Ra (2 μg/ml) was added to the cultures or LP MPs (1×105/well) were
stimulated with recombinant IL-1β (20 ng/ml, Peprotech) for 24 h. After incubation,
cells or supernatants were collected for analysis.
In vivo antibody or antagonist treatment
To deplete neutrophils systemically, mice received intraperitoneal (i.p.) injections of
100 μg of purified monoclonal anti-Ly6G antibody (1A8, Tianjin Sungene Biotech.)
or a control isotype on days -3 and -2 before initiation of DSS drinking, then twice a
week (days 2 and 5 each week) throughout the entire experimental period to ensure
depletion. Neutrophil depletion was evaluated periodically with a hematoxylin and
eosin-stained blood smear and confirmed at the end of each experiment using flow
cytometry.
To block IL-1 signaling, mice were injected i.p. with IL-1 receptor antagonist
(IL-1Ra), kindly provided by Dr. Haiyan Hong (Beijing Proteomics Research Center)
at a dose of 0.5 mg/mouse on days 1, 3, and 5 of every cycle of DSS drinking. To
assess the efficacy of IL-1Ra treatment in late-stage CAC, mice were injected i.p.
with IL-1Ra at the same dose as above daily for 10 days after the last cycle of DSS
drinking (days 84-93) and were sacrificed on day 100.
Tumor transplantation
The mouse colon carcinoma cell line CT26 was kindly provided by Dr. Zhihai Qin
(National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese
Academy of Sciences, Beijing, China) and injected (1×106/mouse) subcutaneously
into one flank of 6-week-old male nude mice (Jackson Laboratory). 2 days after tumor
inoculation, neutrophils (5×104 in 50 μl volume/mouse, viability>98% identified by
trypan blue staining) or the same volume of PBS were injected intratumorally.
Starting 2 days after neutrophil injection, tumor length and width were measured daily
for 10 days with calipers and the tumor size calculated using the formula: tumor
size=0.4 × (length × width2). After sacrifice, the tumors were excised and imaged. In
some settings, neutrophils were incubated with CFSE dye (5 μM) for 20 min and
injected as described above. On day 1, 5, 9 after injection, blood were collected and
processed for FACS analysis as well as tumor tissues were routinely prepared for
frozen section and analyzed by fluorescence microscopy.
Commensal flora depletion
Six-week-old male Balb/c mice were left untreated or were treated with ampicillin (1
g/L), vancomycin (500 mg/L), neomycin sulfate (1 g/L), and metronidazole (1 g/L) in
the drinking water for 4 weeks. During CAC induction, administration of the above
cocktail of antibiotics was started one month after the start of CAC induction (day 28)
and was continued until the mice were sacrificed.
Quantitative RT-PCR
RNA was extracted and reverse-transcribed into cDNA using the SuperScript III First
Strand cDNA synthesis system (Invitrogen). cDNA was synthesized from 0.5 μg of
RNA using random hexamer primers and SuperScript III (Invitrogen). Real-time
RT-PCR was performed on a Bio-Rad iCycler to quantify mRNA levels using the
primers as following:
mIL-6:
forward
5'-
GAGAGGAGACTTCACAGAGGATAC-3';
reverse
5'-GTACTCCAGAAGACCAGAGG-3'
mIL-1β:
forward
5'-GCCCATCCTCTGTGACTCA-3';
reverse
5'-TCTTGGGCTACACTGAGGAC-3';
reverse
5'-AGGCCACAGGTATTTTGTC-3'
mGAPDH:
forward
5'-CATACCAGGAAATGAGCTTGA-3'
hIL-6:
forward
5'-CTGCAGCCACTGGTTCTGT-3';
reverse
5'-CCAGAGCTGTGCAGATGAGT-3'
hIL-1β:
forward
5'-GGAGATTCGTAGCTGGATGC-3';
5'-GAGCTCGCCAGTGAAATGAT-3'
reverse
hGAPDH:
forward
5'-AACGGATTTGGTCGTATTG-3';
reverse
5'-GGAAGATGGTGATGGGATT-3'
All reactions were performed in triplicate. The data were analyzed using Q-Gene
software and expressed as the fold change mean normalized expression (MNE) from
the control value. The MNE is directly proportional to the amount of RNA for the
target gene relative to the amount of RNA for the reference gene, GAPDH.
Flow cytometry
A single cell suspension prepared from colonic tissues as described above was fixed
using PBS containing 1% paraformaldehyde and stained on ice for 25 min with
antibodies in PBS containing 2% heat-inactivated fetal calf serum. The antibodies
used were fluorescein isothiocyanate (FITC)-labeled anti-murine Ly-6G (1A8,
Biolegend), phycoerythrin (PE)-labeled anti-murine CD11b (M1/70, Biolegend), Gr-1
(RB6-8C5, eBioscience), and allophycocyanin-labeled anti-murine CD11b (M1/70,
BD Pharmingen). Data collection and analysis were performed on a FACS Calibur
flow cytometer using CellQuest software (Becton Dickinson). For cell sorting, a
single cell suspension from the spleen or colon was stained with antibodies and sorted
on a FACS AriaII (Becton Dickinson). The antibodies used for sorting were those
described above plus FITC-labeled anti-murine TCR-β (H57-597, eBioscience),
ΥδTCR (UC7-13D5, BD Pharmingen), or CD3 (145-2C11, eBioscience), PE-labeled
anti-murine CD49b (DX5, Biolegend), CD11c (N418, BD Pharmingen), or F4/80
(BM8, Biolegend), or FITC-labeled anti-human CD66b (G10F5, Miltenyi Biotec.) or
CD68 (Y1/82A, BD Pharmingen).
Statistical analysis
Data are presented as the mean±SD. Student’s t test (paired or unpaired) and 1-way
analysis of variance (ANOVA) were used to determine significance, with a P＜0.05
being considered significant. Statistical tests were performed using SPSS 10.0 for
Macintosh, and graphs were prepared using Deltagraph (SPSS, Chicago, I11.).