Supplementary Materials and Methods
Isolation of mouse intestinal epithelial cells and blood mononuclear leukocytes
(ML). Mouse colonic segments were excised and flushed with ice-cold
phosphate-buffered saline (PBS) for the isolation of epithelial cells, as described
previously (1-3). Segments were inverted on an iron rod (diameter ~3 mm) and then
were immersed in PBS with 5.56 mM glucose (PBS-G) containing 0.5 mM
dithioerythritol (DTT) for 30 min. The inverted intestines then were placed in PBS-G
containing 30 mM EDTA for 20–30 min at 37°C with gentle shaking every 5 min.
Epithelial cell purification was achieved by passing the cell suspension through a
mesh filter with a pore size of 40 μm. The cells on the mesh were collected by eluting
the inverted filter with PBS-G containing 1mM EDTA. Cells then were washed again
with PBS-G. The viability and purity of colonocytes were determined in a pilot study
that confirmed trypan blue-negativity (91.7  0.6%), BerEP4 (an epithelial
marker)-positivity (91.1  1.2%), and the absence of CD68 (a monocyte/macrophage
marker) transcripts in the cell preparation. Isolated cells were subjected to RNA
extraction for RT-PCR analysis of CD14, TLR4, and MD2 mRNA.
Heparinized mouse blood was overlaid onto an equal volume of Histopaque
1083, and samples were centrifuged at 400 g for 30 min without brake. After
removing the upper plasma layer, cells at the interface were collected into a fresh
tube and washed three times in ice-cold PBS. Pelleted blood cells (mononuclear
leukocytes) were used for as a positive control in RT-PCR analysis of CD14, TLR4, and
MD2 mRNA.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from samples using Trizol reagent (Life Technologies)
according to the manufacturer’s instructions. The RNA (2 μg) was reverse transcribed
with oligo(dT)15 using RevertAid™ First Strant cDNA Synthesis kit (Thermo) in 20 μL
reaction volume. The resulting cDNA corresponding to 0.1 µg of initial RNA was then
subjected to PCR by the addition of master mix containing 1X PCR buffer, 1 U
DreamTaq™ DNA Polymerase, 0.2 mM dNTPs mixture, 0.4 μM upstream primer, and
0.4 μM downstream primer. The specific primer pairs for PCR reaction were as
follows: human CD14 (forward, 5’-CTGCAACTTCTCCGAACCTC-3’ and reverse,
5’-CCAGTAGCTGAGCAGGAACC-3’),
5’-TGAGCAGTCGTGCTGGTATC-3’
human
MD2
human
and
(forward,
mouse
CD14
(forward,
human
and
reverse,
(forward,
5’-CAGGGCTTTTCTGAGTCGTC-3’),
5’-ATTGGGTCTGCAACTCATCC-3’
5’-CGCTTTGGAAGATTCATGGT-3’),
5’-CCAAGAGCCTCCAGTAGACG-3’
reverse,
TLR4
and
PKCζ
reverse,
(forward,
5’-CCATCCATCCCATCGATAAC-3’),
5’-GTCAGGAACTCTGGCTTTGC-3’
and
reverse,
5’-TGGCTTTTACCCACTGAACC-3’),
mouse
TLR4
(forward,
5’-TGGCATCATCTTCATTGTCC-3’ and reverse, 5’-CGAGGCTTTTCCATCCAATA-3’), mouse
MD2
(forward,
5’-CTTTTCGACGCTGCTTTCTC-3’
5’-ATCCATTGGTTCCCCTCAGT-3’),
mouse
5’-CACCAGCTTACCCCATGACT-3’
mouse
CD68
(forward,
and
reverse,
and
Lgr5
reverse,
(forward,
5’-CTCCTGCTCTAAGGCACCAC-3’),
5’-TTCTGCTGTGGAAATGCAAG-3’
and
reverse,
5’-AGAGGGGCTGGTAGGTTGAT-3’), β-actin (forward, 5’-GGGAAATCGTGCGTGAC-3’
and reverse, 5’-CAAGAAGGAAGGCTGGAA-3’). All of the primer pairs were designed
based on the sequences reported in NCBI nucleotide database. The DNA thermal
cycler was programmed to perform a protocol as follows: 95°C for 3 min for 1 cycle;
95°C for 30 sec (denaturation), 55°C for 30 sec (annealing), and 72°C for 30 sec
(extension) for 30 cycles; and 72°C for 7 min for final extension. Negative controls
were performed with samples lacking cDNA that was not reverse transcribed. RT-PCR
products were then electrophoresed in a 1% agarose gel in the presence of 0.5
μg/mL ethidium bromide, visualized with an ultraviolet transilluminator, and
photographs were taken. The intensity of the DNA bands was analyzed using the
Gel-Pro Analyzer 4.0 software (4).
Histopathological examination. Intestinal tissues were fixed in 4% PFA and
embedded in paraffin wax with proper orientation of the crypt to villus axis before
sectioning. Sections of 5 μm thickness were deparaffinized with xylene and graded
ethanol, stained with haematoxylin and eosin (H&E), and observed under a light
microscope (1, 5).
Immunofluorescent staining and confocal microscopy. The intestinal tissues or cell
monolayers were processed for immunofluorescent staining as previously described
(1, 3, 4). Briefly, intestinal samples are fixed with 4% PFA for 1 h on ice, followed by
OCT solution for 1 h and snap frozen in liquid nitrogen. Tissue sections were
quenched with 50 mM NH4Cl in PBS for 10 min at room temperature. The cells on
filter supports were fixed with 4% PFA, permeabilized with 0.5% Triton X-100 and
quenched with 50 mM NH4Cl. After blocking with 1% BSA, tissue and cells were
stained with primary antibodies to CD14, TLR4, or MD2, followed by secondary
antibodies. The cell nuclei were stained with Hoechst dye. The filter support was cut
off and mounted on slides, and images were captured using a fluorescent
microscope or a laser-scanning confocal microscope (Zeiss LSM780, Germany).
Western blotting. Intestinal mucosal proteins were extracted with complete
radio-immunoprecipitation (RIPA) buffer and subjected to SDS/polyacrylamide gel
electrophoresis (PAGE) (4–13% polyacrylamide) as described (2, 5). The resolved
proteins were then electrotransferred onto PVDF membranes. Blots were blocked
with 5% (w/v) nonfat dry milk or 5% (w/v) bovine serum albumin in Tris-buffered
saline (TBS) containing 0.1% (v/v) Tween 20 (TBS-T) for 1 h, washed with TBS-T, and
incubated with a primary antibody at 4°C overnight. The membrane was washed and
incubated with a secondary antibody for 1 h. After washing, the membranes were
incubated with chemiluminescent solution and signals detected.
Enzyme-linked immunosorbent assay (ELISA). Mouse blood samples were collected
by intracardiac puncture. The levels of Tumor Necrosis Factor (TNF)-alpha (Peprotech)
and Keratinocyte Chemoattractant (KC; murine analog of IL-8) (R&D systems) were
measured using ELISA development kits according to the manufacturer's
instructions.
Quantification of mucosa-associated LPS levels. To measure mucosal LPS levels,
scraped colonic mucosa were homogenized in 99 volumes of pyrogen-free distilled
water and were centrifuged at 3,000 rpm for 15 min. The resultant supernatants
were eluted through nitrocellulose filters (0.45 μm pore size) and were applied to
chromogenic Limulus amoebocyte lysate (LAL) tests using an Endotoxin Quantitation
kit (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s
instructions. The amount of p-Nitroaniline released by endotoxin-activated LAL
enzyme was measured in a spectrophotometer at 405 nm after diazotization.
Cell lines. Human CRC Caco-2 and HT29 cells were grown in Dulbecco’s Modified
Eagle’s Medium (DMEM) (Life Technologies) (4, 6, 7). T84 cells were grown in
DMEM/Nutrient Mixture F-12 Ham (DMEM/F-12) (Sigma). The medium was
supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 mg/ml
streptomycin (Sigma). Human holo-transferrin (11 mg/l; Sigma) and HEPES (15 mM)
were added to the medium for Caco-2 cell culture. Cells were grown to confluence at
37°C with 5% CO2 in a humidified incubator for experiments.
Mouse
leukemic
monocyte/macrophage
RAW264.7
cells
and
human
promonocytic THP-1 cells were grown in RPMI 1640 (Life Technologies)
supplemented with 10% FBS, 10 mM HEPES, 100 U/ml penicillin, and 0.1 mg/ml
streptomycin. The culture medium for RAW264.7 cells contained 0.05 mM
2-mercaptoethanol (Life Technologies). Culture medium was replenished every 2–3 d.
Differentiation of THP-1 cells was induced by treating with 100 ng/ml phorbol
12-myristate 13-acetate (Sigma) for 2 d on culture plates at a concentration of 5106
cells/well. Cells were grown to confluence at 37°C with 5% CO2 in a humidified
incubator for experiments.
Lipid extraction and ultra-performance liquid chromatography (UPLC)-tandem mass
spectrometry (UPLC-MS/MS). Cells were harvested for lipid extraction as previously
described with some modifications (8). Caco-2 cells (3×105 cells) resuspended in 800
μl PBS were mixed with 90 μl of internal standard (C6 ceramide; 90 μl of 1 μg/ml
ethanolic solution) and 3 ml of chloroform/methanol (1:2, v/v), vortexed for 30 s, and
incubated overnight at 4°C. After adding 1 ml chloroform and 1 ml PBS, mixtures
were centrifuged at 2,000 g for 10 min for phase separation. The organic layer was
obtained and evaporated to dryness under nitrogen gas. The dried residue was
dissolved in 100 μl of mobile phase solution (acetonitrile:ammonium formate = 1:1,
v/v).
UPLC-MS/MS was performed using an Acquity UPLC system (Waters, Milford,
MA, USA) coupled with a Quatro Micro triple-quadrupole mass spectrometer
(Micromass, Manchester, UK) equipped with an electrospray ionization source. The
mass spectrometer was operated in the positive ion electrospray mode with selective
reaction monitoring. Analyses were performed in positive mode with a cone voltage
of 20 V for AA I and 20 V for AA II, an extractor voltage of 3.0 V, an RF lens voltage of
0.0 V, a capillary voltage of 3.0 kV, an ion source temperature of 80°C, and a
desolvation temperature of 450°C. A cone gas flow of 50 L/h and a desolvation gas
flow of 1100 L/h were used. Argon (99.99%) was used as a collision gas. The gas flow
rate was kept at 0.13 nl/min in the collision cell. The collision energy was 12 eV for
AA I and 10 eV for AA II. MassLynx 4.1 software was used for data analysis.
Chromatographic separation was performed using an Acquity BEH C18 column (100
mm × 2.1 mm, 1.7 μm) (Waters). The mobile phase was composed of 5 mM
ammonium formate (pH 4.0) (solvent A) and acetonitrile (solvent B). The gradient
profile was as follows: 0–2 min: linear from 50% to 90% B; 2–16 min: linear from 90%
to 100% B; 16–20 min: 100% B; 20–20.1 min: linear from 100% to 50% B; 20.1–23
min: 50% B then re-equilibration of the column. The flow rate was kept at 0.3 ml/min.
The column oven was set at 40°C, and the auto-injection system was set at 4°C. A
partial loop with needle overfill mode was used for sample injection, and the
injection volume was 5 μl.
Plasmid constructs and point mutation. The expression vector pMyc-CMV1-huTLR4
was kindly provided by Dr. Douglas T. Golenbock (University of Massachusetts
Medical School, Worcester, MA, USA). The coding sequences of all plasmid constructs
were confirmed by automated sequencing analysis. Point mutations were created by
site-directed mutagenesis using KAPA HiFi PCR kits (Kapa Biosystems, Woburn, MA,
USA), according to the manufacturer’s instructions. The following primer pairs were
used for Asp299Gly mutation (D299G) (nucleotide change, A896G): forward
(5’-ttagactactacctcgatgGtattattgacttattt-3’)
(5’-aaataagtcaataataCcatcgaggtagtagtctaa-3’);
(nucleotide
change,
and
for
reverse
Thr399Ile
C1196T):
mutation
(T399I)
forward
ctgttctcaaagtgattttgggacaaTcagcctaaagtatttagatctgagc-3’)
and
(5’reverse
(5’-gctcagatctaaatactttaggctgAttgtcccaaaatcactttgagaacag-3’); and for Pro714His
mutation
(P714H)
(nucleotide
(5’-ctacagagactttattcAcggtgtggccattgctgc-3’)
change,
C2141A):
and
forward
reverse
(5’-gcagcaatggccacaccgTgaataaagtctctgtag-3’). Nucleotide changes are shown in
uppercase. Cells were transfected with WT and mutant pMyc-CMV1-huTLR4 plasmids
(2.5 μg/well each) or with pMyc-CMV1 mock plasmids using PLUS Reagent and
Lipofectamine LTX Reagent (Life Technologies) for 24 h. The next day, cells were
trypsinized and reseeded into cell culture plates for 48 h prior to LPS challenge.
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