Reagents and mice

Recombinant murine IFN-γ (CAT# 315-05) and TNF-α (CAT# 315-01A) were purchased from PeproTech (Rocky Hill, USA). Antibodies against GAPDH (CAT# 2118, RRID: AB_561053), NF-κB p65 (CAT# 8242, RRID:AB_10859369), phospho-NF-κB p65 (CAT# 3033, RRID:AB_331284), STAT1 (CAT# 14994, RRID:AB_2737027), phospho-STAT1 (Tyr701) (CAT# 7649, RRID:AB_10950970), IκBα (CAT# 4814, RRID:AB_390781), phospho-IκBα (CAT# 2118) and ubiquitin (CAT# 3936, RRID: RRID:AB_331292) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Antibodies against iNOS (CAT# ab178945, RRID:AB_2861417) and recombinant mouse osteopontin protein (CAT# ab281820) were purchased from Abcam (San Francisco, USA). Antibodies against OPN (CAT# AF808, RRID:AB_2194992) and OPN neutralizing antibodies (CAT# 441-OP) were purchased from R&D Systems (Minnesota, USA). Antibodies against c-Myc (CAT# sc-40, RRID:AB_627268) and c-Myc-HRP (CAT# sc-40-HRP) were from Santa Cruz (California, USA). Antibodies against HA (CAT# 2013819) were purchased from Roche (Natley, New Jersey, USA). Antibodies for Alexa Fluor 488 donkey anti-goat IgG (CAT# A-11055, RRID:AB_2534102) and Alexa Fluor 647 goat anti-rabbit IgG (CAT# A27040, RRID:AB_2536101) were from Thermo Fisher Scientific (Waltham, Massachusetts, USA). L-NMMA (CAT# S7920) and fludarabine (CAT# S1491) were purchased from Selleckchem (Houston, USA). Antibodies against Flag (A8592, RRID:AB_439702), ConA (CAT# L7647) and Griess (CAT# 23479) reagents were purchased from Sigma‒Aldrich (St Louis, MO, USA). Female WT and OPN−/− C57BL/6 mice (8–10 weeks old) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and maintained under specific pathogen-free conditions in the vivarium of Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences. Sample sizes of all experiments were predetermined by calculations derived from our experience. No sample was excluded from the analyses. Animals were not randomly assigned during collection, but the strain, sex, and age of the mice were the same, and the data analysis was single masked. Investigators were not blinded to the group allocation during the experiment and outcome assessment. The number of replicates were indicated in each figure legend. All mice were housed in SPF-rated cages.

Cells

Murine bone marrow MSCs were isolated as previously described [8]. Briefly, murine MSCs were generated from the bone cavity of femurs and tibias of 3- to 4-week-old C57BL/6 mice. The cells were cultured in DMEM-low glucose containing 10% FBS, 2 mM L-glutamine and 1% penicillin‒streptomycin (all from Life Technologies, Carlsbad, California, USA). A single-cell suspension of bone marrow cells was seeded in 100 mm culture dishes, nonadherent cells were removed after 24 h, and the medium was replenished every 2 days. Cells were used at the 9–16th passages. ‘Stemness’ of murine MSCs was determined by their expression of specific cell surface markers and by their capability to differentiate into adipocytes, osteoblasts, and chondrocytes. The MSCs used in each experiment were paired and at the same passage. 293T cells were purchased from ATCC (Manassas, USA).

Quantitative real-time polymerase chain reaction (PCR)

TRIzol (Life Technologies) was added to mice tissues or MSCs for extraction of RNA. The collected tissues should be homogenized with homogenizer. Samples can be stored at − 80 °C before extraction. Total RNA extracted with TRIzol was tested by NanoDrop (Thermo Scientific) for the assessment of RNA quality and quantity. And then RNA was reverse-transcribed into cDNA with a reverse transcription kit from TaKaRa (Tokyo, Japan). Quantitative PCR was performed using FastStart Universal SYBR Green Master Kit (Roche) on an ViiA 7 Real-Time PCR System (Applied Biosystems). The reaction protocol used was 95 °C 5 min, 35 cycles with 95 °C 15 s, 60 °C 60 s, and 72 °C 5 min. β-actin, which was recommended as the reference gene in both software analysis and literature review, was used as an internal control to normalize for differences in the amount of total RNA in each sample. Primers are listed in Table 1.

Table 1 Quantitative real-time PCR primersImmunoblotting and immunoprecipitation

Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Haimen, China) containing phenylmethylsulfonyl fluoride (PMSF) (Beyotime) for 30 min on ice. Lysates were clarified by centrifugation at 15,000 × g for 30 min. The protein concentration of the supernatant fraction was determined by the Bradford assay (Thermo Fisher Scientific, New Hampshire, USA). Protein samples were diluted in 4 × sodium dodecyl sulfate (SDS) loading buffer (TaKaRa), heated to 100 °C for 5 min and fractionated in a 10% SDS–polyacrylamide gel. Proteins were electroblotted onto polyvinylidene fluoride and incubated for 1 h in 5% bovine serum albumin in phosphate-buffered saline (PBS) or nonfat dry milk dissolved in PBS containing 0.1% Tween-20 (PBST) at room temperature. The blotted membranes were incubated with primary antibodies diluted 1000-fold overnight at 4 °C, extensively washed in PBST, incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Boston, USA) diluted 5000-fold for 1 h at room temperature, and washed again with PBST. The blotting membranes were developed with chemiluminescent reagents (Millipore, Billerica, MA, USA) according to the instructions provided by the manufacturer. For immunoprecipitation, the indicated antibody was added to the cell lysates and incubated at 4 °C on a shaker overnight after preclear, followed by pulling down the desired protein with Protein A/G magnetic beads (HY-K0202, Med ChemExpress, New Jersey, USA). The precipitated proteins were boiled with SDS loading buffer and assessed by immunoblotting.

Overexpression and siRNA transfection

Full-length mouse iOPN cDNA was synthesized by Genscript (Nanjing, China). These cDNAs were subcloned into pcDNA3.1 vectors containing an N-terminal Myc epitope tag. Murine MSCs or 293T cells were transfected with pcDNA3.1 or pcDNA3.1 containing iOPN by using Lipofectamine 2000 (Life Technologies). Full-length mouse STAT1-pcDNA3.0 containing Flag was also synthesized. siRNA oligonucleotides were purchased from GenePharma (Shanghai, China) and transfected into mouse MSCs with Lipofectamine RNAiMAX reagent (13,778, Invitrogen, California, USA) following the manufacturer’s protocol. The effects of transfection were tested by immunoblotting or quantitative real-time PCR analysis.

Immunofluorescence

MSCs were seeded into 12-well plates with lysine-coated slides and stimulated with TNF-α and IFN-γ for 24 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% (vol/vol) Triton X-100 (T9284, Sigma‒Aldrich, Missouri, USA), blocked with 2% BSA (sc-2323, Santa Cruz, USA), and incubated with anti-OPN antibody overnight. Cells were washed with PBST and incubated with fluorescent secondary antibody and DAPI. For the OPN and STAT1 colocalization assay, murine MSCs and 293T cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100, followed by blocking with bovine serum albumin (Roche, Basel, Switzerland) at room temperature for 1 h and incubation with specific primary antibodies at 4 °C overnight. After washing with PBS 3 times, secondary antibodies labeled with different fluorescence were added and incubated for 2 h. Confocal microscopy (ZEISS Cell Observer) was used to examine the slides, and images were obtained.

In vitro T-cell proliferation assay

Murine MSCs with different treatments were irradiated with 30 Gy from a 137 Cs source as previously described [8] to inactivate MSCs by inhibiting their proliferation while preserving their immunosuppressive capacity and then seeded into 48-well plates. Freshly isolated splenocytes (4 × 105 cells/well) from C57/BL6 mice were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE, Thermo Fisher Scientific, MA, USA), cocultured with murine MSCs for 3 days in the presence of mouse anti-CD3/CD28 antibodies (eBiosciences, San Diego, CA, USA), and then collected for flow cytometric analysis on a fluorescence-activated cell sorting (FACS) Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Griess assay

Fifty microliters of culture supernatant fraction of different treated murine MSCs and standards were added to a flat bottomed 96-well plate, and then 50 μL of Griess reagent (Sigma) was added. The absorbance at 540 nm was read after incubation for 15 min in the dark at room temperature, and the NO concentrations were calculated.

ConA-induced inflammatory liver injury in mice

Male C57BL/6 mice (8–10 weeks old) were intravenously injected with ConA in PBS at 20 mg/kg to induce inflammatory liver injury. Murine Vector-MSCs (control group) and iOPN-MSCs (1 × 106) were intravenously administered to mice immediately after ConA injection. Isoflurane was used as an inhaled anesthetic at a concentration of 2–3% to induce mice anesthesia 8 h after ConA injection. Blood was sampled from mice eyes. Mice were euthanized by cervical dislocation. Mice serum, spleens and liver tissues were sampled. Liver histological sections fixed in 4% paraformaldehyde were stained with hematoxylin and eosin. Liver MNCs were purified by a Percoll gradient, while spleen MNCs were purified by a Ficoll gradient, and both MNCs were stained with anti-CD4, CD25 and CD69 (eBiosciences) for 30 min at 4 °C in staining buffer and then analyzed by flow cytometry on a FACSCalibur flow cytometer. For T-cell proliferation analysis in vivo, 1 mg BrdU (Cat. No. 559619, BD Biosciences) in 100 μL PBS was intraperitoneally injected into mice. The mice were euthanized 2 h after BrdU administration, and immune cell suspensions from livers or spleens were prepared for flow cytometric analysis.

Inflammatory bowel disease in mice

Male C57BL/6 mice (8–10 weeks old) were given 2.5% (W/V) DSS drinking water, followed by fresh DSS drinking water on Days 3 and 5. Intestinal damage in mice was observed on Days 7 and 9. Murine Vector-MSCs (control group) and iOPN-MSCs (1 × 106) were intravenously administered to mice every 2 days. Mice were euthanized by cervical dislocation, and colon tissues and MLNs were isolated on Day 10. Colon histological sections fixed in 4% paraformaldehyde were stained with hematoxylin and eosin. Colon MNCs and MLNs were purified by a Percoll gradient, and both MNCs were stained with anti-CD4, LY6G and F4/80 (eBiosciences) for 30 min at 4 °C in staining buffer and then analyzed by flow cytometry on a FACS Calibur flow cytometer (BD Biosciences).

TUNEL assay

Collected liver tissues were routinely embedded in OCT compound and then stored at -80 ℃. For the TUNEL assay, apoptotic cells were detected using a TUNEL Apoptosis Detection Kit (Alexa Fluor 488, Cat. No. 40307ES60, YEASEN Biotechnology, Shanghai, China) according to the manufacturer’s instructions.

Induction and detection of murine MSC proliferation and apoptosis

Murine MSCs were treated with IFN-γ plus TNF-α (10 ng/mL) for 24 h. For proliferation analysis, the medium was removed, and the cells were treated with new FBS-free medium followed by the addition of CCK-8 solution (Dojindo Laboratories, Mashikimachi, Kamimashiki gun Kumamoto, Japan). After 2 h, BioTEK (Winooski, VT) was used to record the absorbance at 450 nm. For apoptosis analysis, cells were stained with annexin V and propidium iodide (BD Biosciences) according to the manufacturer’s protocol and then analyzed by flow cytometry.

RNA-sequencing analysis

Total RNA was extracted from TNF-α (10 ng/mL) plus IFN-γ (10 ng/mL)-stimulated or unstimulated MSCs and subjected to RNA-sequencing analysis by Shanghai Biotechnology Corporation Tech Solutions. The Affymetrix Mouse 430 2.0 chip was used in the project. Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 3.1 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US). Significantly differentially expressed genes were acquired by setting a threshold with corrected p < 0.005 and log2 ≤  − 1.5 or log2 ≥ 1.5, and are presented as heatmaps. The correlation analysis was conducted using the R package Hmisc, employing Spearman's rank correlation coefficient. Gene correlation heatmap was plotted by the R package heatmap.

Flow cytometry

For surface marker staining, the collected cell suspensions from mouse tissues were stained with the indicated antibodies for 30 min and subjected to flow cytometry analyses. For the intracellular staining of iNOS, MSCs were fixed and permeabilized by fixation/permeabilization buffer (Cat. No. 00-5523, eBioscience) before staining. An antibody against iNOS (Cell Signaling Technology) was incubated for 30 min, and the secondary antibody labeled with fluorescence was added after washing the cells. MSCs were analyzed by flow cytometry after 30 min.

Chromatin immunoprecipitation (ChIP) assay

Chromatin of MSCs (1 × 107) was prepared using the EZ-ChIP Chromating Immunoprecipitation Kit (Cat. No. 17-371, Millipore, MA, USA) according to the instructions provided by the manufacturer. Chromatin was immunoprecipitated using anti-p-STAT1 or respective IgGs. ChIP-enriched and input DNA were extracted by a PCR Cleanup Kit (Cat. No. AP-PCR-500G, Axygen, California, USA) and analyzed by quantitative real-time PCR, with the inputs as the internal control. Three kinds of primers for the ChIP assay were provided according to the motif as follows: Opn-2667, sense 5′-cctcctcgagttagaagagcgt-3′ and antisense 5′-cattctaactcccaagtgccacac-3′; mouse Opn-2697, sense 5′-ctgctttgcagaattgctccca-3′ and antisense 5′-cattctaactcccaagtgccacac -3′; Opn-2743, sense 5′-gctggtatgagtgtaggaagagggaa-3′ and antisense 5′-cattctaactcccaagtgccacac-3′. Regions of the OPN promoter used for the ChIP assay ranged from − 5000 bp to the TSS.

Statistical analysis

All data are shown as the mean ± SEM from at least three independent experiments. Significant differences were evaluated using one-way ANOVA or the Mann–Whitney U test with GraphPad Prism (version 9.0, GraphPad Software) and Statistical Package for Social Science software (version 22.0, SPSS). Values of p less than 0.05 were considered significant. Sample sizes of all experiments were predetermined by calculations derived from our experience. Test samples were assayed in replicates. To provide measures of reproducibility, three replicates per sample of the assay was considered for our analysis.