CFTR Knockdown induces proinflammatory changes in intestinal epithelial cells

Materials

Eagle’s minimum essential medium, McCoy’s 5a medium, fetal bovine serum (FBS), non-essential
amino acids (NEAA), penicillin-streptomycin (PS), phosphate buffered saline (PBS),
bovine serum albumin (BSA) and puromycin were obtained from Wisent (St-Bruno, Qc,
Canada). Short hairpin RNAs (shRNAs) targeting CFTR and individually cloned into plko.1-puromycin
vector were purchased from Open Biosystems (Huntsville, AL). A lentiviral negative
control, pLKO.1-scrambled, was purchased from Addgene (Cambridge, MA). Hexadimethrine
bromide (Polybrene), forskolin, 3-Isobutyl-1-methylxanthin (IBMX), actinomycin D,
Triton X-100 and paraformaldehyde came from from Sigma (St-Louis, MO). Recombinant
human IL-1? and TNF were from PeproTech (Quebec, Canada). Trizol and M-MLV reverse
transcriptase were from Invitrogen (Carlsbad, CA). The 1X SsoFast EvaGreen Supermix
with Low ROX and the protein assay kit were from Bio-Rad (Hercules, CA). M-PERâ„¢ mammalian
protein extraction buffer was from Thermo Scientific (Rockford, IL). Antibodies used
were from the following suppliers: anti-CFTR and phosphorylated and total anti-p38,
anti-Extracellular signal-regulated kinases (ERK) 1/2, anti-c-Jun N-terminal kinases
(JNK) and anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells
inhibitor alpha (I?B?) were from Cell Signaling Technology (Beverly, MA), anti-Bcl-2-associated
X protein (Bax) and anti-NF-?B p65 were from Santa Cruz Biotechnology Inc. (Dallas,
TX), anti-?-actin was from Sigma and species-specific horseradish peroxidase (HRP)-conjugated
secondary antibodies were from Bio-Rad. Alexa Fluor® 594 donkey anti-goat (1:1000)
and the ProLong® Gold antifade reagent with 4?,6-diamidino-2-phenylindole (DAPI) were
from Life Technologies Inc. (ON, Canada). The Amershamâ„¢ ECLâ„¢ Prime Western Blotting
Detection Reagent was from GE Lifesciences (Baie d’Urfe, QC, Canada). Western-blot
stripping buffer was from ZmTech Scientifique (Montreal, QC, Canada).

Intestinal cell lines

The human colon carcinoma cell line, Caco-2/15, a stable clone from the parent Caco-2
cells (American Type Culture Collection, Rockville, MD), was obtained from Dr. Emile
Levy (CHU Sainte-Justine Research Center, Montreal, Quebec, Canada). HT-29 cells,
a human colon carcinoma cell line, were purchased from ATCC®. Although of cancerous
origin, these cells are widely used for the study of non-malignant epithelial intestinal
physiology. Caco-2/15 were used for all experiments and maintained at subconfluent
stages in EMEM supplemented with 5 % FBS, 1 % NEAA and 1 % PS. All experiments were
carried out on cells from passage 26 to 31. HT-29 cells were maintained at subconfluent
stages in McCoy’s 5a supplemented with 10 % FBS and 1 % PS and specific experiments
were carried out on cells from passage 7 to 9. Cells were seeded onto 12-well plates
at a density of 2?x?10
⁵
cells/well after a trypan blue exclusion test to assess cell survival. Cells were
cultured for a period of three to four days prior to any experimentation.

Lentivirus production and cell infection

Lentiviral stocks were prepared with the use of HEK293FT as packaging cells according
to the method described previously 14], 15]. The viral supernatants were collected 2 days after, and lentiviruses concentrated
following a fast ultrafiltration with Amicon® Ultra-15 centrifugal filter device (EMD
Millipore, Bellerica, MA). Concentrated lentiviruses were aliquoted and stored at
?80 °C until use.

Caco-2/15 and HT-29 cells were infected with specific lentivirus in the presence of
Polybrene as published previously 14], 15]. When cells reached 80 % confluence, they were plated and allowed to proliferate
in the presence of puromycin to select for cells showing stable integration of the
shRNA constructs. Infection efficiency was assessed by measuring CFTR gene and protein
expression by quantitative PCR (Q-PCR) and Western Blotting. Cells infected with lentivirus
containing scrambled shRNA sequence served as controls since preliminary experiments
had shown comparable cell viability and integrity as well as gene and protein CFTR
expression between scrambled and non-infected cells 14].

Pharmacological activation of CFTR

To activate CFTR, cells were treated 24 h with a cAMP-increasing cocktail made of
10 ?M forskolin and 100 ?M 3-Isobutyl-1-methylxanthin (IBMX) in the absence of serum.

Proinflammatory challenge and cell viability

To induce a proinflammatory state, subconfluent (80–90 % of confluence) cells, corresponding
to homogenously undifferentiated cells, were stimulated with 10 or 25 ng/mL of recombinant
human IL-1? or TNF for times ranging from 5 minutes to 24 h. Trypan blue staining
and cell counting were performed to determine whether the compounds had any effect
on cell viability. All experiments were carried out in the absence of serum except
for experiments with HT-29 cells, as these cells exhibited high cell mortality in
the absence of serum.

Interleukin-6, ?8 and ?10 secretion

Cell culture supernatants were collected, centrifuged for 5 min at 13 000 rpm at 4 °C
to remove cell debris and stored at ?80 °C until they were assayed for IL-6, ?8 and
?10 by ELISA (OptEIA antibody set; BD Biosciences, ON, Canada), according to the manufacturer’s
instructions. Absorbances were measured at 450 nm wavelength using the Spectra RainBow
plate reader (Tecan Systems inc., San Jose, CA). Cellular protein concentration was
determined using the Bio-Rad protein assay kit and concentration of interleukins normalized
to total protein content.

RNA isolation, RT-PCR and Q-PCR

RNA was isolated from cells using Trizol according to manufacturer’s instructions.
Two ?g of total RNA was reverse transcribed by using the M-MLV reverse transcriptase.
CFTR and IL-8 mRNA expressions were determined by Q-PCR. Briefly, 15 ?l Q-PCR reaction
containing cDNA diluted 100 times, 1X SsoFast EvaGreen Supermix with Low ROX and 0.5 ?M
of CFTR or IL-8 specific and intron-spanning primers was performed using the Mx3000p
Q-PCR System (Agilent Technologies, Santa Clara, CA) under the following conditions:
95 °C for 30 s, 45 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 20 s. Each
reaction was performed in duplicate. The relative quantification of both genes was
normalized to the 60S ribosomal protein L27 (RPL27) reference gene and fold-induction
was determined from the average threshold cycle (Ct) using the standard curve method.
PCR efficiency was calculated from the slope of the standard curve, and generated
using a five-fold dilution series of cDNA template obtained from IL-1?-treated cells.

IL-8 mRNA stability

IL-8 mRNA stability was assessed by decay after the addition of actinomycin D. Briefly,
CFTR knockdown and control cells were treated first, as detailed above, then transcription
was blocked by the addition of 5 ?g/mL of actinomycin D. Cells were collected, at
times ranging from 2 to 8 h after actinomycin treatment and total RNA extracted using
Trizol. Q-PCR for IL-8 was carried out as described and IL-8 mRNA levels were normalized
to RPL27, whose expression remained unaffected by actinomycin treatment.

Immunoblotting

Cells were washed twice with cold PBS 1X, scraped, and lysed with cold M-PERâ„¢ mammalian
protein extraction buffer. Cell lysates were pelleted through centrifugation at 13
000 rpm at 4 °C for 5 min. Lysates were stored at ?20 °C for later use. Protein concentration
was determined using the Bio-Rad protein assay kit. Briefly, between 15 to 40 ug of
total protein were separated on 10 % SDS-PAGE and transblotted onto polyvinylidene
difluoride (PVDF) membranes (GE Lifesciences). After blocking for 1 h in a TBS solution
mixed with 5 % nonfat dehydrated milk, membranes were blotted overnight at 4 °C with
anti-CFTR (1:1000), or phosphorylated anti-p38, anti-ERK 1/2, anti-JNK, anti- I?B?
and anti-Bax (all 1:1000) followed by a 1 h-incubation with species-specific horseradish
peroxidase (HRP)-conjugated secondary antibodies (1:10,000). Visualization of the
protein bands on X-ray film (Bioflex MSI film; Ultident, St-Laurent, QC, Canada) was
achieved with the Amershamâ„¢ ECLâ„¢ Prime Western Blotting Detection Reagent. In some
cases, membranes were stripped with a western-blot stripping buffer according to the
manufacturer’s instructions, and reprobed overnight at 4 °C with anti-?-actin (1:5000),
or with total p38, ERK 1/2, JNK, and I?B? (1:1000). Before protein visualization,
membranes underwent a second 1 h-incubation with species-specific HRP-conjugated secondary
antibodies (1:20,000). The films were quantified by computer-assisted scanning densitometry
using UN-SCAN-IT software (Silk Scientific, Orem, UT).

NF-?B immunofluorescence

To determine the expression of NF-?B, 4?x?10
⁴
Caco-2/15 cells were grown on glass Lab-Tek®II chamber slides™ (Fisher Scientific,
ON, Canada) 2 to 3 days before experiments. Cells were then washed with PBS 1X and
fixed with paraformaldehyde 3.7 % at room temperature for 10 min. After two PBS washes,
cells were permeabilized with Triton 0.1 % at room temperature for 10 min and washed
again with PBS. Cells were then blocked with a BSA 1 % solution at room temperature
for 30 min before being labeled with a goat anti-human NF-?B p65 antibody (1:200)
for 1 h at room temperature. After 3 washes with the blocking solution, cells were
incubated with an Alexa Fluor® 594 donkey anti-goat IgG (1:1000) for 1 h at room temperature.
Cells were then washed 3 times with the blocking solution and then twice again with
PBS after which the media chambers were removed. Slides were mounted with the ProLong®
Gold antifade reagent with DAPI for nucleus staining and the immunofluorescence was
detected using a fluorescent microscope (Leica Microsystems Inc., ON, Canada). The
level of fluorescence in a given region (nucleus, whole cell and background) was quantified
with the Image J program using the area, the integrated density and mean gray value
readings. Corrected total cell fluorescence (CTCF) was calculated using the following
equation: CTCF?=?Integrated Density – (Area of selected region x Mean gray value of
background reading). The cytoplasmic CTCF was obtained after subtracting the nucleus
CTCF from its whole corresponding cell CTCF.

Mice

The Cftr?/? and Cftr +/+ mice on a BALB/c background were initially provided by Dr. Christina Haston from
McGill University, Montreal, Canada. All mice were bred and housed in the CHU Sainte-Justine
Research Institute animal facility. To avoid risk of intestinal obstruction and premature
death, all Cftr ?/? mice were fed ad libitum standard chow diet moistened with reverse osmotic water and 17.8 mmol/L polyethylene
glycol 3350 (PEGLYTEâ„¢; Pharmascience, Quebec, Canada) in their drinking water from
three weeks of age until euthanasia. To eliminate the confounding effect of diet on
intestinal physiology, control mice were also maintained on the same feeding and drinking
regimen. These regimens did not alter food and water consumption of the mice. After
weaning, Cftr?/? and Cftr +/+ mice were grouped based on their sex and were co-housed. Mice were euthanized at
12 weeks of age by intracardiac puncture under anesthesia. All procedures were in
accordance with the CHU Sainte-Justine Research Institute Animal Care committee.

Histology

At sacrifice, the entire intestinal tract was collected and inspected to detect the
presence of intestinal obstruction; none of the mice had visual evidence of such obstruction.
The entire jejunum, which corresponds roughly to the second third of the small intestine,
was removed, longitudinally opened, gently flushed with ice-cold PBS and cleared of
feces. The specimens were fixed with buffered-formalin phosphate 10 %, paraffin-4 ?m
sections were prepared, mounted on microscope slides, and stained with hematoxylin
eosin safran. Eosinophils were quantified in intercryptic lamina propria by counting
at 400 X magnification the number of cells in ten randomly selected fields. Female
and male mice were analysed separately by a pathologist blinded to the genotype and
sex of the mice.

Statistical analysis

Data are expressed as means?±?SEM. Comparison of means was achieved using analysis
of variance, unpaired Student’s t or nonparametric Mann–Whitney tests where appropriate. All statistical analyses were
conducted with SPSS 21 software. Statistical significance was set at p?0.05.