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SUPPLEMENTARY MATERIALS AND METHODS
Histological examination and immunohistochemistry
Colons were removed from mice and fixed in 10% neutral-buffered formalin solution
and then embedded in paraffin, cun into tissue sections, and stained with hematoxylin
and eosin.
Paraffin-embedded slides were deparaffinized and immersed in 80℃ water bath
in 10 mM sodium citrate buffer with 0.1% Tween 20 overnight for antigen unmasking.
Slides were incubated with primary antibody against C3 (Hycult Biotech.), Ki67
(Dako) or Gr-1 (BD Biosciences) in PBS containing 1% BSA and 10% goat serum.
Biotinylated secondary antibody (Dako) were added and incubated at room
temperature for 1 hr. Streptavidin-HRP (BD Pharmingen) was added, and after 40 min
the sections were stained with DAB substrate and counterstained with hematoxylin.
Detection and quantitation of apoptotic cells
For detection of apoptotic cells in tissues, labeling of degraded DNA specific to
apoptotic
cells
was
performed
using
a
modification
of
the
terminal
deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end-labelling
(TUNEL) technique by application of in-situ cell death detection kits (Roche
Diagnostics), according to the manufacturer’s instructions. Levels of apoptosis were
quantified by counting the number of TUNEL-positive cells per 100 nuclei. The
apoptotic index was obtained from the ratio of apoptotic to total cells.
Colon homogenates
A 1 cm segment was divided from the distal 4 cm of the harvest colon. Wet weight
was recorded separately for the whole distal 4 cm and the portion taken for
homogenation. Colon tissue samples were homogenized in PBS containing a cocktail
of protease inhibitors (1 μl to 20 mg of tissue according to the manufacturer’s
protocol, Roche Diagnostics) with a Polytron homogenizer and centrifuged at 12 000
g for 10 min. the supernatants were stored at -20℃ until used for ELISA analysis.
Whole colon culture
200-300 mg of The colon tissues on the border of sigmoid colon and rectum was
dissected and washed in cold PBS supplemented with penicillin and streptomycin.
These segments were cut into small pieces (0.5×0.5 cm) and cultured (three pieces
per mouse) in 24-well flat bottom culture plates in serum-free RPMI1640 medium at
37℃ for 24 hr. Supernatants were centrifuged at 13,000 rpm at 4℃ for 5 min and
stored at -80℃ until use.
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, 30
μg of the supernatants were separated on 10% SDS-polyacrylamide gel and
transferred onto an Immunobilon-P Transfer membrane (Millipore). After being
blocked with 5% skim milk, the membrane was incubated with primary antibodies
at1:1000 dilution. Rabbit anti-actin antibody was used as an internal control
(purchased from Tianjin Sungene Biotech.). ImmunoPure peroxidase-conjugated
anti-rabbit IgG were used as secondary antibodies. The blotted membrane was then
treated with the Super Signal West Dura Extended Duration Substrate (Pierce) and
signals were detected by LAS-3000 mini CCD camera (Fuji Film). Primary antibodies
used for immunoblotting were purchased from Cell Signaling.
Generation of bone marrow chimeric mice
Cell suspensions from male WT, C3-/- or C5aR-/- bone marrow (BM) were prepared
from femurs and tibias, filtered, and counted. Recipient male mice had been lethally
irradiated with 900 rads (2 doses of 450 rads with a 3-h resting peroid) from a cobalt
source. Six hours after irradiation, recipient irradiated mice received 1×107 BM cells
via the tail vein. Genomic DNA was extracted from blood and bone marrow
chimerism was determined 4 weeks later by PCR for C3 or C5aR, and systemic C3
was assayed by flow cytometry via zymosan binding followed by staining.
Isolation of mouse 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 spleen cells, mouse lamina propria immune cells, and in vitro
treatment
A splenic single cell suspension was obtained by crushing the spleens through a 200
m mesh nylon filter. To isolate 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 myeloid cells were then isolated by sorting
for CD11b+ cells and neutrophils were isolated 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 6 hr with 50nM recombinant mouse C5a (R&D
systems) or lipopolysaccharide (LPS, 100 ng/ml; Sigma-Aldrich). In some cases,
neutrophils were cocultured for 24 hr with LP myeloid cells from normal mucosa at a
ratio of 1:2. To assess a direct role of IL-1 signaling in driving IL-17A expression by
LP myeloid cells, IL-1Ra (2 μg/ml) was added to the cultures or LP myeloid cells
(1×105/well) were stimulated with recombinant IL-1β (20 ng/ml, Peprotech) or plus
IL-23 (100 ng/ml, Peprotech) for 24 hr. After incubation, cells or supernatants were
collected for analysis. In addition, to determine a role of MAPK ERK signaling
pathway, neutrophils (5×105/well) were seeded and stimulated with 0-100 nM
recombinant C5a for 15 min. Otherwise, at the indicated time points following
incubation, cells were collected for immunoblotting examination. In some settings,
cells were preincubated with MEK1/2 inhibitor (U0126, 10 μM) or NF-κB inhibitor
(Bay11-7082, 20 μM) for 30 min, followed by C5a stimulation. After incubation, cells
were collected for analysis.
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 Gr-1 (RB6-8C5,
eBioscience),
IL-17A
(eBio17B7,
eBioscience),
phycoerythrin
(PE)-labeled
anti-murine TCR-β (H57-597, eBioscience), ΥδTCR (UC7-13D5, BD Pharmingen),
RORγt (AFKJS-9, eBioscience), and allophycocyanin-labeled anti-murine CD11b
(M1/70, BD Pharmingen), CD3 (145-2C11, eBioscience). For intracellular cytokine
staining, cells were restimulated with PMA (50ng/ml)+ionomycin (500ng/ml) plus
brefeldin A (dilution at 1:1000) for 4 hours, then cell-surface antigen was stained
routinely. After the last wash, cells were fixed and permeabilized by using
intracellular fixation&permeabilization buffer (eBioscience). 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 described above and sorted on a FACS AriaII (Becton
Dickinson).
Quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR)
RNA extracted from colon epithelial cells or cell lines was reverse-transcribed into
cDNA using the SuperScript Ⅲ First Strand cDNA synthesis system (Invitrogen).
cDNA was synthesized from 0.5 μg RNA using random hexamer primers and
SuperScriptⅢ(Invitrogen). Real-time RT-PCR was performed on a Bio-Rad iCycler
to quantify mRNA levels. The primers for real-time were listed in supplemental Table.
All reactions were performed in triplicate. The data were analyzed using Q-Gene
software and expressed as fold change mean normalized expression (MNE) from
control value. MNE is directly proportional to the amount of RNA of the target gene
relative to the amount of RNA of the reference gene, GAPDH.