Mice and reagents
C57BL/6 (B6) wild type (WT) mice were purchased from Jung Ang Lab Animal, Inc. (Seoul, Korea). The IL4/GFP (4get) and IFNγ/YFP (yeti) cytokine reporter mice were kindly provided by Dr. R. Locksley (University of California at San Francisco, CA, USA). Foxp3/GFP reporter B6 mice and CD11c-diphtheria toxin receptor (DTR) transgenic (tg) mice were obtained from Dr. Rho H. Seong and Dr. E. Choi, respectively (Seoul National University, Seoul, Korea). All of the mice were on a B6 genetic background, were maintained at Sejong University, and were used for experiments at 6–12 weeks of age. They were maintained on a 12-h light/12-h dark cycle in a temperature-controlled barrier facility with free access to food and water. These mice were fed with a γ-irradiated sterile diet and autoclaved tap water. In this study, age- and sex-matched mice were used for all the experiments. The animal experiments were approved by the Institutional Animal Care and Use Committee at Sejong University (SJ-20130802). Our experiments were conducted in a blinded and randomized trial. For the induction of colitis, DSS with a molecular weight of 36–50 kDa was purchased from MP Biomedicals (Solon, OH, USA). LPS derived from Escherichia coli (serotype 0111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Preparation of TWE
Taheebo, the dried inner bark of Tabebuia avellanedae Lorentz ex Griseb., was purchased from Frontier Natural Products Co-op. (Norway, IA, USA). To prepare the TWE, five hundred grams of taheebo was stirred in 5 L cold water at 4 °C for 24 h. After filtration, the solution was reduced to 10% of the original volume with a 40 °C rotary evaporator under vacuum. The extracts were freeze-dried, ground to a fine powder, and stored at 4 °C until use.
Standardization of TWE by HPLC
TWE was standardized with 6-O-(3,4-dimethoxybenzoyl)-ajugol and 6-O-(p-dimethoxybenzoyl)-ajugol known as constituents in Tabebuia avellanedae Lorentz ex Griseb. [12], using high-performance liquid chromatography (HPLC; SCL-10A, Shimadzu, Japan) installed with a UV-VIS detector (SPD-10A; Shimadzu, Japan). Sample filtration was performed using membrane filter (Maidstone, Kent, UK) with pore size 0.45 μm prior to injection. Sunfire C18 column (4.6 mm × 250 mm; Waters, USA) was used in reversed-phase chromatography. The temperature within the chamber was maintained at 30 °C. A 10 μl aliquot of the sample was injected onto a C18 reversed-phase column and the absorbance was detected at 260 nm. Sample concentration was calculated from a standard calibration curve obtained with 6-O-(3,4-dimethoxybenzoyl)-ajugol and 6-O-(p-dimethoxybenzoyl)-ajugol. 6-O-(3,4-dimethoxybenzoyl)-ajugol and 6-O-(p-dimethoxybenzoyl)-ajugol were found in TWE at a mean level of 12.29 mg/g and 16.25 mg/g, respectively.
Flow Cytometry
The following monoclonal antibodies (mAbs) from BD Bioscience were used: fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-Cy7, or allophycocyanin (APC)-conjugated anti-CD3ε (clone 145-2C11); PE- or APC-conjugated anti-CD11c (clone HL3); PE-conjugated anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) (clone UC10-4F10–11); PE-Cy7-conjugated anti-CD11b (clone M1/70); APC-conjugated anti-F4/80 (clone BM8); PE-Cy7- or APC-conjugated anti-CD4 (RM4–5); APC-conjugated anti-CD25 (clone PC61); PE-conjugated anti-MHC II (clone M5/114.15.2); biotin-conjugated anti-CD86 (clone GL1); PE-conjugated anti-tumor necrosis factor α (TNFα) (clone MP6-XT22); PE-conjugated anti-IL6 (clone MP5-20F3); PE-conjugated anti-IL10 (clone JES5-16E3); PE-conjugated anti-IL12p40 (clone C15.6); and PE-conjugated anti-IFNγ (clone XMG1.2). The following mAbs were obtained from eBioscience (San Diego, USA): FITC- or PE-conjugated anti-IL4 (clone BVD6-24G2); FITC- or PE-conjugated anti-IL17A (clone eBio17B7); PE-conjugated anti-inducible nitric oxide synthase (iNOS) (clone CXNFT); and FITC- or PE-conjugated anti-Foxp3 (clone NRRF-30). The following mAb from R&D Systems was used: PE-conjugated anti-arginase-1. To perform surface staining, the cells were harvested and washed twice with 0.5% BSA-containing cold PBS (FACS buffer). To block the Fc receptors, the cells were incubated with anti-CD16/CD32 mAbs on ice for 10 min and were subsequently stained with fluorescence-labeled mAbs.
Intracellular cytokine staining
To perform intracellular staining, splenocytes were incubated with brefeldin A, an intracellular protein transport inhibitor (10 μg/ml), in RPMI medium for 2 h at 37 °C. The cells were stained for cell surface markers, fixed with 1% paraformaldehyde, washed once with cold FACS buffer, and permeabilized with 0.5% saponin. The permeabilized cells were then stained for an additional 30 min at room temperature with the indicated mAbs (FITC-conjugated anti-IL17 or anti-IL4; PE-conjugated anti-IFNγ, anti-IL4, anti-IL10, anti-IL6, anti-IL12, anti-TNFα, anti-iNOS, or anti-arginase-1; FITC- or PE-conjugated isotype control rat IgG mAbs). Fixation and permeabilization were performed using a Foxp3 staining kit (eBioscience) with the indicated mAbs (FITC-conjugated anti-Foxp3; PE-conjugated anti-Foxp3; or FITC- or PE-conjugated isotype control rat IgG mAbs). More than 5000 cells per sample were acquired using a FACSCalibur, and the data were analyzed using the FlowJo software package (Tree Star, Ashland, OR, USA).
Cell isolation by magnetic activated cell sorting (MACS) and culture
Splenic CD4+ T cells were isolated from mice using a MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer’s instructions. The purity of CD4+ T cells was >97% after MACS. Mesenteric lymph nodes (MLNs) were aseptically removed, and single-cell suspensions of the MLN were obtained by homogenization and passing through a 70 μm nylon cell strainer. Primary macrophages and the RAW264.7 macrophage cell line were cultured in RPMI 1640 (Gibco BRL, USA) culture media supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 units/mL penicillin-streptomycin, and 5 mM 2-mercaptoethanol.
In vivo depletion of DCs
In order to deplete CD11c+ DC populations, CD11c-DTR tg recipient mice were intraperitoneally injected with diphtheria toxin (DT) (120 ng/mouse) twice 3 days apart and subsequently mice were sacrificed for experiments 2 days after the second injection.
Determination of cell viability
To examine the effect of TWE on cell viability of RAW264.7 cells, RAW264.7 cells were seeded in 24-well plates at a cell density of 1 × 106 cells/ml (2 × 105 cells/well) and stimulated with either TWE (100, 300, 900, and 2700 μg/ml) or LPS (1 μg/ml) for 12 h at 37 °C in a CO2 incubator. Total cells were washed in PBS, and 100 μl annexin V buffer (BD Pharmingen, San Diego, CA) containing 10 μl 7-amino actinomycin (7-AAD) was added for 15 min at room temperature in the dark. Cells were resuspended in 300 μl annexin V buffer and were analyzed within 1 h by flow cytometry. The relative cell viability (%) was expressed as a percentage (7-AAD negative) relative to the control cells.
Polarization of macrophages with M1 or M2 stimuli
To examine the effect of TWE on the polarization towards the M2 phenotype, RAW264.7 cells (1 × 106 cells) were seeded in 24-well plates and stimulated with either TWE (100, 300, and 900 μg/ml) or vehicle in the presence of IL4 (20 ng/ml) for 12 h at 37 °C in a CO2 incubator. In addition, to test the effect of TWE on M1 polarization, RAW264.7 cells were stimulated with either TWE (100, 300, and 900 μg/ml) or vehicle in the presence of IFNγ (100 ng/ml) for 12 h at 37 °C in a CO2 incubator. LPS (1 μg/ml) was used as positive control in the same culture conditions as above. After treatment, the polarization status of the RAW264.7 cells was determined by evaluating the expression of either iNOS for M1 or arginase-1 for M2 by flow cytometry.
Induction of colonic inflammation
Before DSS treatment, two groups of B6 mice were fed with either TWE-containing or control water for 5 days. Mice in the TWE-treating group were fed with approximately 2 mg of TWE per day. Subsequently, these mice were fed with 3% (w/v) DSS in either TWE-containing or control water ad libitum for 5 days. After 5 days of DSS administration, both groups of mice were given normal control water for 5 days until sacrifice for experiments. To evaluate the clinical symptoms of DSS-induced colitis, the mice were monitored for a change in the percentage of body weight (0, none; 1, 1–10%; 2, 11–20%; 3, >20%), stool consistency (0, normal; 1, loose stool; 2, diarrhea), and bleeding (0, normal; 1, hemoccult positive; 2, gross bleeding) on a daily basis during colitis induction for 10 days. The body weight was expressed as a percentage of weight change for each individual mouse and was calculated relative to the starting body weight on day 0. These data were used to calculate a disease activity index (DAI).
Histology
Distal colonic sections were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 6 μm sections using a microtome (RM 2235, Leica, Germany). The sections were then stained with H&E for the analysis of histological changes. The histological score of each individual mouse was measured as follows: epithelial damage (E), 0 = none; 1 = minimal loss of goblet cells; 2 = extensive loss of goblet cells; 3 = minimal loss of crypts and extensive loss of goblet cells; 4 = extensive loss of crypts; and infiltration (I), 0 = no infiltrate; 1 = infiltrate around the crypt basis; 2 = infiltrate reaching the muscularis mucosa; 3 = extensive infiltration reaching the muscularis mucosa and thickening of the mucosa with abundant edema; 4 = infiltration of the submucosa. The total histological score was calculated as E + I.
Statistical analysis
Statistical significance was determined using the Excel software (Microsoft, USA). To compare two groups, Student’s t-test was performed. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered significant.