Transforming Growth Factor β1/SMAD Signaling Pathway Activation Protects the Intestinal Epithelium from Clostridium difficile Toxin A-Induced Damage

Cell line culture and toxin A.Rat intestinal jejunal crypt cells (IEC-6) were obtained from Banco de Células do Rio de Janeiro (BCRJ) and cultured in Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12; Gibco Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 5 mg of bovine insulin (Sigma-Aldrich), 50 IU/ml of penicillin, and 100 μg/ml of streptomycin (Invitrogen Molecular Probes) at 37°C in a 5% CO₂ incubator. For in vitro experiments, toxin A (TcdA) from Clostridium difficile was purchased from Sigma-Aldrich (St. Louis, MO; C3977-2UG). For in vivo experiments, TcdA proteins were obtained from supernatants of C. difficile strain VPI 10463 grown in a dialysis system culture and purified as described previously (44). Native TcdA was purified by anion-exchange chromatography, followed by gel filtration chromatography. The purity of the toxins was determined by SDS-PAGE and mass spectrometry.

Immunocytochemistry.Immunostaining was performed, as described previously (45), to investigate the effect of TcdA on TGFRII and phosphorylated SMAD2/3 (pSMAD2/3) distribution in IEC-6 cells. Briefly, the cells were seeded overnight at 3 × 10⁵ cells/well in a 24-well plate. Subconfluent cultures were left untreated (control) or incubated with different treatments: (i) 10 ng/ml TcdA, (ii) TGF-β1 (10 ng/ml), or (iii) TcdA plus TGF-β1 (both at 10 ng/ml) in standard DMEM/F-12. The concentration of toxin A was chosen based on previous data (24) indicating that preincubation of T84 cells with concentrations equal to or less than 10 ng/ml of TcdA increased the release of TGF-β1. Additionally, Brito and colleagues (32) showed that 100 ng/ml of TcdA induced death in IEC-6 cells characteristic of apoptosis and necrosis, and a concentration of 10 ng/ml induced death in a lower percentage of cells. After 24 h, cells were fixed for 15 min with 4% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.6), washed with PBS, and permeabilized with 0.1% Triton X-100 for 5 min. Samples were then blocked for 1 h with PBS containing 5% bovine serum albumin (BSA) and incubated for 1 h at room temperature with the following primary antibodies: rabbit anti-phospho-SMAD2/3 (1:50; Santa Cruz Biotechnology) and rabbit anti-TGRII (1:100; Santa Cruz Biotechnology). Samples were then incubated for 1 h at room temperature with anti-rabbit secondary antibodies conjugated with Alexa Fluor 488 (1:400; Invitrogen Molecular Probes) and/or Alexa Fluor 546 (1:1,000; Invitrogen Molecular Probes). Slides were mounted with Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Images were captured using a Nikon Eclypse TE2000-S fluorescence optical microscope. The total number of cells in each random field was quantified by counting DAPI-stained nuclei, and the percentage of cells stained for the different markers in each random field was determined. To analyze TGF-β1/SMAD pathway activation, number of cells with SMAD2/3 nuclear translocation was quantified and expressed as the percentage of total cells (measured by DAPI staining). At least 5 fields per slide in 3 slides per condition were analyzed. At least three independent experiments were performed. Levels of TGFRII in IEC-6 cells were quantified by measuring the intensity of TGFRII immunostaining in IEC-6 cell cultures. To do that, TGFRII immunostaining was quantified under different experimental conditions, using integrated density values generated with the ImageJ program (National Institutes of Health, USA). Arbitrary values for labeling intensity were expressed in relation to the number of cells in the field (DAPI staining). At least 5 fields were analyzed per condition and at least three independent experiments were performed.

Proliferation assay.Ki67 is a nuclear protein that is tightly linked to the cell cycle and is a marker of cell proliferation. Proliferation was performed as described previously (49). Briefly, the cells were seeded overnight at 3 × 10⁵/well in a 24-well plate. Subconfluent cultures were left untreated (control) or incubated with different treatments: (i) 10 ng/ml of TcdA, (ii) TGF-β1 (10 ng/ml), or (iii) TcdA plus TGF-β1 (both at 10 ng/ml) in standard DMEM/F-12. After 24 h, cells were fixed for 15 min with 4% paraformaldehyde in PBS (pH 7.6), washed with PBS, and permeabilized with 0.1% Triton X-100 for 5 min. Samples were then blocked for 1 h with PBS containing 5% bovine serum albumin and incubated for 1 h at room temperature with the following primary antibody: rabbit anti-Ki67 (Santa Cruz Biotechnology). After wash, the slides were incubated with Dako-labeled polymer (Envision Flex no. K4010; Dako) for 1 h. Slides were mounted, and Ki67 samples were stained with the chromogen 3,3-diaminobenzidine (DAB; Dako), counterstained with Mayer's hematoxylin, and then mounted in Faramount mounting medium (Dako). Images were captured using a Leica DM 2000. Cells positive for Ki67 were counted in 10 fields per slide in 4 slides per treatment. At least three independent experiments were performed.

Flow cytometric analysis of apoptosis and necrosis.Apoptosis and necrosis were assessed by flow cytometry. The ApoAlert annexin V kit (BDR Biotech) was used to detect phosphatidylserine on the reverse membrane surface of apoptotic cells. Propidium iodide was used to detect nucleic acids inside necrotic and late apoptotic cells. IEC-6 cells were seeded in 6-well plates at a concentration of 10⁶/well and incubated for 24 h. Then the cells were treated as described above for an additional 24 h. After this period, cells were washed twice with PBS, trypsinized (0.05% trypsin; Sigma-Aldrich), collected by centrifugation, and doubly stained with fluorescein isothiocyanate-conjugated annexin V and propidium iodide (both from BDR Biotech). The samples were then analyzed on a Coulter EPICS XL flow cytometer (EasyCyte; Guava Technologies).

Scratch wound assay.IEC-6 cells were seeded in 6-well plates at a concentration of 10⁶/well and allowed to grow until total confluence. The confluent cells were scratched with a razor blade at the midline, extending to the right side of the well, as described elsewhere (26). After the scrape or “wounding,” the wells were washed with PBS twice, and the medium was changed to fresh medium containing TcdA (10 ng/ml), TGF-β1 (10 ng/ml), or TcdA (10 ng/ml) plus TGF-β1 (10 ng/ml). The control cells were incubated with medium alone. Migrated cells were observed under a magnification of ×100 after 12 and 24 h using an Olympus 1 × 71 inverted microscope with a QImaging camera. Images were acquired using QCapture Pro.5.1 software, and cells were counted in 10 fields/well.

Animals.C57BL/6 mice, weighing 25 to 30 g, were housed in temperature-controlled rooms under 12-h light-dark cycles. The animals received water and food ad libitum. Surgical procedures and animal treatments were conducted in accordance with the guidelines for institutional and animal care and use of the Federal University of Ceará, Brazil. All procedures involving animals were approved by the Committee on the Ethical Treatment of Research Animals of the Federal University of Ceará.

Induction of intestinal inflammation.The TcdA-induced enteritis mouse model was used as described previously (46). Briefly, mice were fasted overnight but with free access to water. Prior to surgery, mice were anesthetized with ketamine and xylazine (100 mg/kg and 10 mg/kg intramuscularly, respectively). Through a midline laparotomy, a 4-cm ileal loop was ligated and injected with either 0.1 ml of PBS (pH 7.4; control) or PBS containing TcdA (10 μg). The abdomen was sutured, and the animals were allowed to regain consciousness. Four hours after administration of TcdA or PBS, the mice were sacrificed. Intestinal loops were removed and the loop length and weight were measured. The intestinal loop tissues were processed accordingly for analysis using different techniques, including immunohistochemistry, Western blotting, and quantitative real-time reverse transcription-PCR (qPCR).

Immunohistochemistry.Immunohistochemistry assays to evaluate TGF-β1, TGRII, and pSMAD2/3 distribution were performed with the ileal tissues using the avidin-biotin-peroxidase method (47) in formalin-fixed, paraffin-embedded tissue sections (4 μm thick) mounted on poly-l-lysine-coated microscope slides. Briefly, the sections were deparaffinized and rehydrated through xylene and graded alcohols. Afterward, sections were blocked twice with endogenous peroxidase (10 min each) with 3% (vol/vol) hydrogen peroxide and washed in PBS. Sections were incubated overnight at 4°C with rabbit anti-TGF-β1 antibody (1:100; R&D Systems), rabbit anti-TGRII (1:100; Santa Cruz), and rabbit anti-pSMAD2/3 (1:50; Santa Cruz Biotechnology). All the antibodies were diluted in PBS plus BSA. After a washing, the slides were incubated with avidin-biotin-horseradish peroxidase conjugated for 30 min, according to the protocol of the manufacturer (Santa Cruz Biotechnology). TGF-β1, TGFRII, and pSMAD2/3 were visualized with DAB after 2 min of incubation. Negative controls were obtained by omitting primary antibodies. Slides were counterstained, dehydrated in a graded alcohol series, cleared in xylene, and mounted in Faramount mounting medium (Dako Cytomation).

qPCR.Quantitative real-time PCR (qPCR) analysis of mRNA for TGF-β1 and TGFRII was performed for IEC-6 cells and mouse ileal tissue. For culture assays, IEC-6 cells were seeded overnight at 6 × 10⁵/well in a six-well plate, and subconfluent cultures were incubated with or without 10 ng/ml of TcdA in standard DMEM/F-12 medium for 24 h. The ileal tissues were treated with TcdA or PBS as described previously. The RNA was extracted using TRIzol (Invitrogen, USA) according to the protocol provided by the manufacturer. The quality of the RNA was analyzed by a 260/280 ratio and quantified by UV absorption using a NanoDrop ND-1000 (Thermo Fisher Scientific). Two micrograms of total RNA was reverse transcribed with a high-capacity cDNA reverse transcription kit according to the manufacturer's instructions (Applied Biosystems). The primers used in this assay were as follows: for TGF-β1, TAC CAT GCC AAC TTC TGT CTG GG A (forward)/ATG TTG GAC AAC TGC TCC ACC TTG (reverse), and for TGRII, ACT GTC CAC TTG CGA CAA CCA GA A (forward)/AGA AGC GGC ATC TTC CAG AGT GAA (reverse). The following β-actin primers were used as an endogenous control: TGG ATC GGT TCC ATC CTG G (forward)/GCA GCT CAG TAA CAG TCC GCC TAG A (reverse). qPCR was performed using a SYBR green PCR master mix, including Ampli Taq Gold polymerase (Applied Biosystems). Reactions were performed on an ABI Prism 7500 real-time PCR system (Applied Biosystems). The relative expression levels of genes were calculated using the threshold cycle (2^−ΔΔCT) method (48). The amount of target genes expressed in a sample was normalized to the average of the endogenous control.

Immunoblotting assays.Ileal tissue samples (100 mg) were homogenized in lysis buffer plus protease inhibitor tablet (Sigma-Aldrich). The protein concentration was determined using the Bradford assay. Sample proteins (50 μg) were loaded onto a range of polyacrylamide gels (8 to 15%) for electrophoresis and transferred onto nitrocellulose membrane by electroblotting under wet conditions (Mini Trans blot; Bio-Rad). The membranes were incubated overnight at 4°C individually with the following antibodies: anti-TGF-β1/2 and anti-α-tubulin (Santa Cruz Biotechnology) at dilutions of 1:200 and 1:1,000, respectively. The membranes were then incubated with secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology) for 2 h at room temperature at a 1:1,000 dilution. For measurement, the chemiluminescence system was visualized using the ChemiDocTM XRS+ system (Bio-Rad, Life Technologies), and the resulting bands were analyzed and quantified by ImageJ (National Institutes of Health). The α-tubulin gene was used as the housekeeping gene.

Statistical analysis.All data are expressed as means ± standard errors of the means (SEM). The difference between multiple groups were evaluated by one-way analysis of variances (ANOVA) followed by Bonferroni's test using GraphPad Prism (GraphPad, San Diego, CA). Student's t test was used to compare the means of two groups. A P value of <0.05 was considered to indicate significant differences. All experiments were done in triplicate, and all results represent means from at least 3 independent experiments.