Female C57BL/6 mice (9~10 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). All animal experiments have been approved by the Institutional Animal Care and Use Committees of the Catholic University of Korea (Seoul, Korea).
Priming of murine BM-derived MSCs
The BM-derived MSCs were isolated from C57BL/6 mice and expanded, as described in our previous work (19). In brief, BM cells were flushed out from femurs and tibias, plated in 75 cm2 tissue culture flasks at a concentration of 1×106 cells/mL in the complete culture medium, and incubated at 37℃ and 5% CO2. Non-adherent cells were removed after three days, and the remaining cells were passaged into a new flask when the cells reached 70~80% confluency. To do priming, we harvested cells at the 90% confluency and plated them in 12-well plates (5×104 cells/ well) in the complete culture medium supplemented with recombinant mouse IFN-γ (100 ng/mL, R&D Systems, Minneapolis, MN, USA). Poly(I:C) (TLR3 ligand, 10 μg/mL, Sigma-Aldrich, St Louis, MO, USA) was added to the culture medium for stimulation. The primed MSCs were collected after 24 h and used for in vitro and in vivo experiments.
DSS-induced murine colitis and MSC treatments
Experimental colitis was induced by administration of 2.5% DSS (molecular weight 36,000~50,000; MP Biomedicals, Santa Ana, CA, USA) in drinking water ad libitum for five days (day 0~5). Nine-week-old mice were randomly allocated into one of three groups: DSS only (DSS control), DSS with unstimulated MSCs treatment (DSS + MSCs), and DSS with the treatment of MSCs stimulated with IFN-γ and poly(I:C) (DSS + primed MSCs). Unstimulated MSCs or the primed (stimulated) MSCs (3x106 cells) were injected intraperitoneally (i.p.) on days 1 and 3. All mice were sacrificed on day 9 to harvest the spleen, mesenteric lymph node (mLN), and colon tissue. We daily assessed the severity of colitis until day 9 using body weight and the disease activity index (DAI), which is the summation of three parameters (1–4 score for each): body weight loss, stool consistency and occult bleeding (20).
To prepare L-1-methyl tryptophan (L-1MT, Sigma-Aldrich, St. Louis, MO) for oral gavage, 1 g of L-1MT was added to a 15-ml conical tube with 7.8 ml Methocel/Tween [0.5% Tween 80/ 0.5% Methylcellulose (v/v in water; both from Sigma-Aldrich)]. The following day, the L-1MT concentration was adjusted to 85 mg/ml by adding an additional 4 ml Methocel/Tween and mixing again. For in vitro use, L-1MT was prepared as a 100 mmol/L stock in 0.1 N NaOH, adjusted to pH 7.4 and stored at -20°C protected from light.
Hematoxylin-eosin (H&E) staining and Immunohistochemistry (IHC)
We subjected Formalin-fixed, paraffin-embedded tissue sections to H&E staining for microscopic examination. Slides were scored by a pathologist (blinded to experimental group). Pathologic severity of IBD was assessed by eight parameters: inflammatory infiltrate, goblet cell loss, crypt hyperplasia, crypt density, muscle thickness, submucosal infiltration, ulcerations and crypt abscesses (0-3 score for each). A total histological severity score, ranging from 0 to 24, was obtained by summing the eight item scores (21).
Tissue sections (4μm) were mounted on super frost glass sliders and deparaffinized in xylene and a graded series of ethanol, followed by antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Nonspecific binding sites were saturated by exposure to 10% normal goat serum diluted in phosphate buffered saline (PBS) for 60 min. We incubated slides overnight at 4℃ with primary antibodies against mouse Ki-67 (1:100 dilution, Abcam, CB, UK), Lysozyme (1:250 dilution, Abcam), IDO-1 (1: 400 dilution, Biolegend, San Diego, CA), then washed with PBS for 10 min. Biotinylated goat anti-rabbit IgG and rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) secondary antibodies were applied to tissue sections, and the slides were incubated at room temperature for 30 min. After the sections were washed and incubated for 30 minutes with peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark) at room temperature, 3,3’-diaminobenzidine was added to visualize antigens. Sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted. We prepared negative control tissue samples in the same manner as described above, except that the primary antibody was omitted or replaced with an isotype control antibody (R&D Systems, Minneapolis, MN). IHC stains were evaluated for the presence of positively stained cells in 5 random fields under X200 magnification on a light microscope (Leica DMI5000B, Germany). We counted the positively stained cells in each crypt.
We isolated total RNA from colon homogenates with Trizol® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed into cDNA. We did quantitative assessment of target mRNA levels by quantitative RT-PCR using a CFX96TM SYBR Green real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The quantity of mRNA was calculated using the 2–DDCt method, and the level of b-actin was used to normalize total RNA quantities. The sequences of forward and reverse primers are shown in Table 1.
Protein extractions and measurements of cytokines by ELISA
Colon samples were homogenized in 1 ml buffer solution (1 x PBS, 1% NP40, 0.05% Na-deoxycholate, 0.1% SDS, and 1 tablet of Complete Protease Inhibitor Cocktail [Roche Diagnostics, Basel, Switzerland]), centrifuged at 3,000 rpm for 20 min, after which supernatants were harvested. Total protein concentrations in supernatant were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Concentrations of IL-1β, TNF-α, MCP-1, IL-6, and IL-10 were determined by ELISA using a commercially available kits (R&D systems, Minneapolis, MN, USA). Absorbance at 450 nm was measured using a microplate spectrophotometer, Benchmark Plus (Bio-Rad, Richmond, CA, USA).
Flow cytometric analysis
Single-cell suspensions were stained in fluorescence-activated cell sorting (FACS) buffer at 4°C for 30 min. We analyzed samples using an LSRII flow cytometer (BD Pharmingen, San Diego, CA). The following antibodies against mouse antigens were purchased from BD Pharmingen (San Diego, CA): BV605-conjugated anti-CD11b, FITC-conjugated anti-CD11c, BV450-conjugated anti-CD4, FITC-conjugated CD25, and PE-conjugated anti-Foxp3.
We did the following experiments to analyze the effect of MSCs on T-cell proliferation. We isolated untouched T cells from splenocytes using the Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). We cocultured 2 x 105 splenocytes with or without MSCs (primed or unstimulated) in the presence or absence of 2 mg/ml anti-CD3/CD28 (ebioscience, San Diego, CA, USA) plus 10 ng/ml recombinant murine TGF-β (R&D Systems) and 50 ng/ml recombinant murine IL-2 (R&D Systems) for 72 hours. Then, T cells were harvested and surface stained for CD4, CD25, and Foxp3. A competitive IDO1 inhibitor, L-1MT (Sigma-Aldrich, St. Louis, MO, USA) was added to some wells.
We performed statistical analyses using GraphPad Prism 7 software (GraphPad Software, Inc., La Jolla, CA, USA). All values are expressed as mean ± standard error of the mean. We did statistical comparisons between groups using the one-way ANOVA test with Bonferroni correction (Post-Hoc). Differences were considered significant when the P value was less than 0.05.