Cell culture
Frozen human UC-MSCs at passage 3 were provided by Konya biotechnology Co., Ltd (Nanjing, China) and maintained in Dulbecco’s modifed Eagle’s medium (DMEM)/F12 (BI, Cromwell, CT, USA) supplied with 10% fetal bovine serum (FBS) (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA), 100 µg/mL streptomycin/penicillin and 2 mM L-glutamine (BI, Cromwell, CT, USA) at 37 ℃ and 5% CO2.
Human fibroblast-like synoviocytes (FLS) were isolated from synovial tissue of RA patients as previously described [27], which was approved by the biomedical ethics committee of Anhui Medical University. Hyperplastic synovium biopsies from RA patients were washed and minced in DMEM (BI, Cromwell, CT, USA) with penicillin/streptomycin. Then, dissected synovial tissues were digested with 0.1 mg/mL of deoxyribonuclease Ⅰ (Sigma) and 1 mg/mL of collagenase Ⅳ (Sigma) and incubated at 37 ℃ for 2 h. Single cells were obtained after filtering and washing, then cultured in DMEM with 20% FBS and 100 µg/mL streptomycin/penicillin at 37 ℃ and 5% CO2. At confluence, adherent cells were trypsinized, and recultured in fresh medium at a ratio of 1:2. Passage 5 was used in this experiment.
Lentiviral vectors construction
Homo cDNAs of sTNFRⅡ (GenBank: BT019927.1, 1-261 encoded amino acids) and Fc domain of immunoglobulin G1 (IgG1) (GenBank: AF027159.2) were synthesized by GeneChem (Shanghai, China). The sTNFRⅡ-Fc fusion gene sequence was generated by combining the two segments with a flexible linker (5’-GGAGGTGGAGGATCA-3’). A total of 150 ng plasmid template was used to amplify and identify sTNFRⅡ-Fc with PrimeSTAR® HS DNA polymerase (Takara, Japan). The amplification program was as follows: 95 ℃ for 5 min, followed by 30 cycles of 98 ℃ for 10 s, 55 ℃ for 10 s, 72 ℃ for 90 s and then 1 cycle of 72 ℃ for 8 min. Primers used for the amplification of sTNFRⅡ-Fc were as follows: 5’-GAGGATCCCCGGGTACCGGTCGCCACCATGGCGCCCGTCGCCGTCTGG-3’ (forward) and 5’-TCCTTGTAGTCCATACCTTTACCCGGAGACAGGGAGAGGCTC-3’ (reverse). Amplified sTNFRⅡ-Fc was then scanned with a Tanon-2500 gel imager system (Tanon, Shanghai, China)
The expression cassette of sTNFRⅡ-Fc was cloned into the lentiviral vector GV358 (GeneChem, Shanghai, China) which contains the enhanced green fluorescent protein (EGFP) and puromycin resistance gene with restriction enzymes AgeⅠ (N-terminus) and BamHⅠ (C-terminus), and the sequence was confirmed by DNA sequence analysis. The resulting lentiviral vector, LV-sTNFRⅡ-Fc, was then cotransfected together with pHelper1.0 (containing rev, pol and gag gene of human immunodeficiency virus) and pHelper2.0 (containing a herpes simplex virus VSV-G gene) into 293T cells using Lipofectamine 2000 Reagent (Thermo Fisher Scientific). Forty-eight hours after transfection, the virus-containing supernatants were collected, filtered (0.45-µm filters), and then concentrated by ultracentrifugation (25000 rpm, 120 min, 4 ℃). After centrifugation, the pellet was resuspended by the virus preservation solution (GeneChem, Shanghai, China), and the transducing unit titer (TU/ml) of lentivirus was determined by quantitative polymerase chain reaction (qPCR) [28].
Generation of sTNFRⅡ-MSC stable cell line
Approximately 1×105 UC-MSCs were seeded into six-well plate, and transfection was performed when UC-MSCs were reached 60% confluent. Culture medium was replaced with 1.5 mL of fresh medium containing lentivirus at a multiplicity of infection (MOI) of 7 pfu per cell with 10 µg/mL of polybrene (Sigma). The virus-containing medium was removed after 12 h transfection, and fresh DMEM/F12 medium containing 10% FBS was added. The cells were cultured at 37 ℃ with 5% CO2 for another 48 h, and then transferred to a 25-cm2 culture flask with 5 mL of fresh medium. Stable cell lines were generated by screening in 625 ng/mL of puromycin (Puro) (Sigma) for 2 weeks, and maintained in 300 ng/ml of puro, then used for experiments within the following 3 weeks. The infection efficiency was determined by flow cytometry and fluorescence microscope.
Concentration of sTNFRⅡ-Fc in supernatants
To detect the level of sTNFRⅡ-Fc in supernatants, 2×105 MSCs or transduced MSCs (including EGFP-MSC and sTNFRⅡ-MSC) at passage 1 or 3 (after transfection) were cultured in six-well plates, and supernatants were collected at varying time-points. Concentrations of sTNFRⅡ-Fc were determined by a human sTNFRⅡ ELISA kit (R&D System, Minneapolis, MN) according to the instructions.
Phenotype of sTNFRⅡ-MSC
Surface antigens of sTNFRⅡ-MSCs were analyzed by flow cytometry. Cells were trypsinized using 0.25% trypsin-EDTA (WISENT, Quebec, Canada), and incubated with 1% bovine serum albumin (BSA; Gibco) for 0.5 h to block non-specific antigen binding. Then, the cells were incubated with PE/APC-conjugated mouse anti-human CD90, CD73, CD105, CD45, CD34 and HLA-DR (Biolegend, USA) respectively for 1 h at 4 ℃.The cells were washed twice and suspended for flow cytometry.
Tri-lineage mesenchymal differentiation assay
sTNFRⅡ-MSCs were tested for multi-lineage differentiation potential using MSCgo™ differentiation kits (BI, Cromwell, CT, USA) for osteogenic, chondrogenic, and adipogenic differentiation according to the instructions. For evaluating the osteogenic ability of sTNFRⅡ-MSCs, cells were fixed and stained with 2% alizarin red S (Sigma-Aldrich, MO, USA), and optical density (OD) of the eluent was measured at 550 nm after eluting with 10% cetylpyridinium chloride (Sigma) for 1 h at room temperature. Alcian blue staining for aggrecan (AGG) was used to assess the chondrogenic ability of sTNFRⅡ-MSCs. Cells were fixed and stained with 1% alcian blue (Sigma) solution overnight at room temperature, and then de-stained with 8 M guanidine hydrochloride (Sigma) overnight at 4 ℃. The guanidine hydrochloride solutions were collected for the measurement of absorbance at 600 nm. For adipogenic differentiation, lipid vesicles were stained with oil red O for 30 min at room temperature, and then eluted using isopropanol for 1 h at room temperature. The absorbance of the isopropanol eluent was detected at 500 nm.
Preparation of conditioned medium (CM)
Briefly, sTNFRⅡ-MSC, EGFP-MSCs or MSCs at passage 5 were cultured with 5% CO2 and at 37 ℃ until 70–80% confluency. Then, fresh DMEM/F12 with less FBS (3%) was replaced to reduce the generation of toxins or uncalled proteins. The supernatant was collected after 48 h culture and filtrated using a 0.22-µm pore filter to remove the cell debris. The collected TNFRⅡ-MSC-CM, EGFP-MSC-CM or MSC-CM were stored at -80 ℃ and used for subsequent coculture procedure.
In vitro functionality of sTNFRⅡ-MSC
Surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 on MSCs culturing with sTNFRⅡ-MSC-CM
Glass coverslips were installed onto the bottom of 24-well plates before MSCs (5×103 cells) were seeded, and cultured for another 12 h. TNFRⅡ-MSC-CM, EGFP-MSC-CM or MSC-CM plus with 20 ng/mL TNF-α (PeproTech, Rocky Hill, NJ, USA) were added into each well and incubated for 48 h. The cells were washed, fixed with 4% paraformaldehyde and blocked with 0.5% BSA. Then, ICAM-1 (Santa Cruz, 1:100) and VCAM-1 (Santa Cruz, 1:50) antibody were added and incubated overnight at 4 ℃. Anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 were used as the secondary antibody. Glass coverslips from each wells were taken out and fluorescent images were collected using a TCS SP8 confocal microscope (Leica, Wetzlar, Germany)
Expression of ICAM-1 and VCAM-1 on sTNFRⅡ-MSC
A total of 2×105 of sTNFRⅡ-MSC, EGFP-MSCs or MSCs were stimulated with TNF-α (20 ng/mL) and/or IL-1β (PeproTech, 20 ng/mL) for 48 h. The expression of ICAM-1 and VCAM-1 on RA-FLS was determined by Western blotting. Briefly, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) for 30 min on ice and then centrifuged at 12000 rpm for 30 min at 4 ℃. Supernatant of the lysates was mixed with SDS-PAGE (Beyotime, China) at the ratio of 5:1, then electrophoretically separated and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were incubated with ICAM-1 (1:200) and VCAM-1 (1:100) antibody respectively overnight at 4 ℃. The dilution of corresponding secondary anti-rabbit/mouse antibody is 1:10,000. Membranes were then scanned with ImageQuant LAS 4000mini (GE Healthcare) and protein bands were quantified using ImageJ software (National Institutes of Health).
Receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) level in coculture system
A 0.4-µm transwell (Costar) was used to coculture RA-FLS and sTNFRⅡ-MSC at the ratio of 2:1. RA-FLS (6×104 cells) were seeded in the six-well plates and cultured overnight until adherence. Then, cells were washed with serum-free DMEM/F12, and 3×104 of sTNFRⅡ-MSC or EGFP-MSC suspensions were added into the upper chamber of the transwell. Meanwhile, TNF-α (20 ng/mL) or IL-1β (20 ng/mL) was added in the coculture system and incubated for 48 h. Culture supernatant was collected for detection of RANKL (Arigo Biolaboratories Corp) and OPG (R&D Systems) levels using commercial ELISA kits according to the manufacturer’s protocol.
In vivo functionality of sTNFRⅡ-MSC
As previously described, lipopolysaccharide (LPS)-induced cytokine storm was aroused in NOD/SCID mice [13, 29]. Briefly, LPS (Sigma) was dissolved in sterile water to a concentration of 3 mg/mL. One week before LPS challenge, mice were pretreated with sTNFRⅡ-MSC (i.v., 5×106 cells), EGFP-MSC (i.v., 5×106 cells) and Etanercept (s.c., 4 mg/kg) respectively on Day 0 and Day 4 as shown in Fig. 2J. Then, mice received a sublethal dosage of LPS (20 mg/kg) via intraperitoneal injection on Day 7. Blood samples were harvested at different time points (0 h, 1.5 h and 6 h after LPS challenge), and centrifugated at 4 ℃, 1000×g, 10 min. Serum was collected and stored at -80 ℃ for quantification of sTNFRⅡ, TNF-α, IL-1β and IL-6 levels using ELISA kits according to the instructions.
Collagen-induced arthritis (CIA) model establishment, treatment and assessment
Collagen-induced arthritis was induced in 8-week-old male DBA/1 mice in conformity to experimental animal ethics committee of Anhui Medical University. Briefly, chick type Ⅱ collagen (CⅡ) (Chondrex, Redmond, WA, USA) was dissolved in 0.1 M glacial acetic acid overnight at 4 ℃. An equal volume of complete Freund’s adjuvant was mixed with CⅡ solution to produce a final concentration of 2 mg/mL emulsion. To induce CIA, DBA/1 mice were injected intradermally at the back and the base of the tail with 100 µl of the prepared emulsion, followed by a booster immunization 21 days later. The day of the first immunization was defined as day 0. After the onset of arthritis (day 28), the mice received a single intravenous injection of 1×106 sTNFRⅡ-MSCs or non-engineered MSC in 150 µl saline by tail vein. Etanercept was served as positive control. Mice were administered 5 mg/kg of etanercept (Guojian Pharmaceutical Co., Ltd., Shanghai, China) three times per week for 3 weeks after the booster immunization. The mice in normal and CIA group were given an equal volume of saline.
Body weight, arthritis index (AI) and swollen joint count (SCJ) were monitored every three days as reported [30]. For AI (16-point scale): 0 = normal; 1 = redness and/or swelling of the paw or one digit; 2, redness and/or swelling of two paws and joints; 3 = more than two joints involved; and 4 = All paws and digits involved with severe arthritis. Regarding SJC (24-point scale): every swollen phalanx joints or ankle joints is counted as 1 point, and thus, the maximum SJC score for each mouse was 24. Animals were sacrificed on day 49, blood and relevant organs were collected for further investigation.
Histological evaluation of the ankle joints and spleens
The hind ankle joints and spleens of mice from each group were removed, washed and fixed in 4% paraformaldehyde at room temperature for 48 h. Regarding hind ankle joints, 10% EDTA (w/v) was used to decalcify the calcium from bone or cartilage. The samples were embedded in paraffin and sections (5-mm) were stain with H&E, Toluidine Blue (Sigma) and Alcian Blue & Alizarin red S (Sigma) respectively. Pathological changes in the spleen and ankles were evaluated by two blinded observers. Established scoring systems were as follows [30]: for ankle joints (5-point scale, 0 = no effect to 4 = severe effect), five parameters were involved, including inflammation, cartilage erosion, hyperplasia of synovium, mononuclear cells infiltration and formation of pannus; regarding spleens (4-point scale, 0 = no effect to 3 = severe effect), five parameters were involved, including the number of germinal centers (GCs), cellularity of the periarteriolar lymphoid sheath (PALS), lymphoid follicles, marginal zone, and red pulp hyperemia.
Detection of thymus index and spleen index
The thymus and spleen were removed aseptically and placed in the centrifuge tubes. The weight of spleens and thymus were collected using a precision electronic scale and the thymus index or spleen index was shown as the ratios of thymus or spleen weight to mouse body weight (milligrams per gram).
T- and B-cell proliferation assay
T cells were isolated from thymus whereas B cells were isolated from spleen of each mouse using lymphocyte separation medium (Dakewe Biotech). 1×106 cells in 100 µl RPMI 1640 (5% FBS) were seeded into 96-well flat-bottom plates. Then, 3 mg/L concanavalin A (ConA, Sigma) and 4 mg/L lipopolysaccharide (LPS, Sigma) were respectively added to stimulate T and B lymphocytes proliferation. Cells were cultured for another 48 h at 37 ℃ with 5% CO2. one hour before the end of culture, A total of 10 µl Cell Counting Kit-8 (CCK-8) (Kumamoto) was added to each well. Then, the absorbance was read at 450 nm.
For collagen Ⅱ-induced specific CD4+ T-cell proliferation, CD4+ T cells of spleen of mice from each group were isolated using a cell sorter (BD FACSAria) [30]. Purified splenic CD4+ T cells (2×105) in 100 µl RPMI 1640 (5% FBS) were seed into 96-well flat-bottom plates and then loaded with 20 µg/ml collagen Ⅱ (Chondrex). After culture for 48 h, 10 µl of CCK-8 was added to each well and then incubated at 37 ℃ with 5% CO2 for two hours. Subsequently, the absorbance was read at 450 nm.
To evaluate the effects of apoptosis/autophagy on the immunosuppressive ability of MSCs, 2×105 of CD4+ T cells isolated from spleen of CIA mice as described above were directly cocultured with MSCs or sTNFRⅡ-MSC in 100 µl RPMI 1640 (5% FBS) at the ratio of 1:1. Before coculture, MSCs or sTNFRⅡ-MSC were incubated with 20 ng/mL TNF-α + 10 µg/mL cycloheximide (CHX, Sigma) (sensitizes cells to TNF-α-induced apoptosis) for 12 h to indue apoptosis and autophagy, and 3-MA (5mM, Sigma) were used as inhibitor of autophagy [31]. After coculture for 48 h, the suspended cells were transferred into a new 96-well plate with 100 µl serum-free RPMI 1640, and10 µl of CCK-8 was added to each well, then incubated at 37 ℃ with 5% CO2 for two hours. Subsequently, the absorbance was read at 450 nm.
Analysis of T- and B-cell subsets in CIA mice
Splenic single cell suspension was harvested after incubation with FACS™ Lysing Solution (BD Pharmingen) for 10 min at room temperature. A total of 1×106 cells in 250 µl PBS were stained with fluorescence-conjugated anti-mouse monoclonal antibodies against CD4, CD25, CD19, CD138, CXCR5 and PD-1 (BD) at 4 ℃ for 30 min. For intracellular staining, the cells were stimulated with Leukocyte Activation Cocktail (BD Pharmingen) for 5 h and then fixed and permeabilized for 30 min using the Fixation/Permeabilization Kit (eBioscience). The permeabilized cells were incubated with fluorescence-conjugated anti-mouse IFN-γ, IL-2, IL-10, IL-17 and Foxp3 for 30 min at 4 ℃ before analyzed by flow cytometry (Cytoflex, Beckman Coulter).
Analysis of cytokines and immunoglobulins in serum
The concentrations of cytokines (TNF-α, IFN-γ, IL-4, IL-10 and IL-17) and immunoglobulins (IgG1, IgG2a and anti-CⅡ) were detected using commercial ELISA kits according to the manufacturers’ instructions.
Immunohistochemistry
Ankle joints immunostaining was conducted to visualize the expression of collagen Ⅱ, MMP-13, tissue inhibitors of metalloproteinase (TIMP)-1 and a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTS)-5 on cartilage and synovium respectively. Sections were incubated with primary antibodies against collagen Ⅱ (1:150 dilution, Abcam), MMP-13 (1:200 dilution, Novus), TIMP-1 (1:100 dilution, Abcam) or ADAMTS-5 (1:100 dilution, Novus) at 4 ℃ overnight and enzyme-labeled goat anti-mouse/rabbit IgG polymer (ZSGB-BIO, Beijing, China) was used as the secondary antibody. Sections were then visualized with 3,3’-diaminobenzidine. The percentages of positive cells or immunostaining intensity was used to assess the expression of relative proteins on joint cartilage and synovium. For cartilage, percentages of MMP-13, TIMP-1 and ADAMTS-5 positive chondrocytes were determined by counting the number of stained cells and dividing by the total number of chondrocytes, whereas collagen Ⅱ expression was counted by immunostaining intensity. For synovium, immunostaining intensity was used to evaluate the abundance of MMP-13, TIMP-1 and ADAMTS-5.
Immunofluorescence assay
CIA mice received 1×106 of EGFP-MSC or sTNFRⅡ-MSC transplantation via tail vein, and were sacrificed 1, 3, 7 and 14 days after injection respectively. The spleens and ankle joints were harvested and fixed in pre-cooled 4% paraformaldehyde for 12 h. After washing with PBS, samples were embedded using optimal cutting temperature compound (OCT). Sections (6-µm) were incubated with anti-human CD105 (1:100, Abcam) at 4 ℃ overnight. The second antibody was anti-human Alexa Fluor 594 for 1 h at 37 ℃. After DAPI staining, sections were observed with a fluorescence microscope (Leica, Wetzlar, Germany).
For detection of apoptosis and autophagy of sTNFRⅡ-MSC in vivo, spleen sections (d14) were incubated with anti-human CD105 (1:100) and cleaved caspase 3 (1:150, Abcam)/LC3B-Ⅱ (1:200, Abcam). The second antibody was anti-human Alexa Fluor 594 for 1 h at 37 ℃. After DAPI staining, sections were observed with a TCS SP8 confocal microscope (Leica, Wetzlar, Germany).
Apoptosis detection
MSCs apoptosis was induced by 20 ng/mL TNF-α and 10 µg/mL CHX (sensitizes cells to TNF-α-induced apoptosis) at different time points (6 h, 12 h and 24 h) [31]. For comparison of anti-apoptosis effect of MSC, EGFP-MSC and sTNFRⅡ-MSC, MSCs were divided into three groups: control group (cultured with MSC-CM), EGFP-MSC group (cultured with EGFP-MSC-CM) and sTNFRⅡ-MSC group (cultured with sTNFRⅡ-MSC-CM). Cells were treated with 20 ng/mL TNF-α and 10 µg/mL CHX for 12 h. At the end of the culture, cells were collected and stained with the combination of annexin V-FITC and propidium iodide (PI)-PE (Annexin V-FITC/PI Apoptosis Detection Kit, BestBio, China) for 30 min. Apoptosis rates were detected by flow cytometry (Cytoflex, Beckman Coulter) and representative fluorescent images were collected by a imaging flow cytometer (ImageStreamX Mark Ⅱ).
Western Blot Analyses
MSCs were treated with 20 ng/mL TNF-α and 10 µg/mL CHX at different time points (6 h, 12 h and 24 h). For comparison of anti-apoptosis effect of MSC, EGFP-MSC and sTNFRⅡ-MSC, cells were treated with 20 ng/mL TNF-α and 10 µg/mL CHX for 12 h. Cell total protein was harvested after lysing and centrifuging at 12000×g for 15 min at 4 ℃. Supernatant was collected and added with 5× protein loading buffer (Beyotime Biotechnology, China), then the protein samples were denatured at 100 ℃ for 10 min. The denatured protein samples were separated by polyacrylamide gel electrophoresis and transferred electrophoretic ally to a polyvinylidene fluoride membrane (Millipore, MA, USA). The dilution of primary antibody of Bcl-2, Bax, Caspase 3, Cleaved Caspase 3, Caspase 8, Cleaved Caspase 8, LC3B Ⅰ/Ⅱ, TRIB3 and β-actin (all from Proteintech, USA) is 1:800. The dilution of second antibody of goat anti-rabbit/mouse is 1:10000 (ZSGB-BIO, Beijing, China). The membranes were scanned with an ImageQuant LAS 4000mini (GE Healthcare) and the density of protein bands was analyzed by ImageJ software (National Institutes of Health).
Immunogenicity detection
2×105 of MSCs were cultured with sTNFRⅡ-MSC-CM, EGFP-MSC-CM and MSC-CM respectively in the presence of 20 ng/mL TNF-α + 20 ng/mL IFN-γ. After culturing for 48 h at 37 ℃ and 5% CO2, cells were collected and stained with fluorescence-conjugated anti-human monoclonal antibodies against HLA-DR for 30 min at 4 ℃. Then, cells were washed twice and analyzed by flow cytometry (Cytoflex, Beckman Coulter).
Chondrogenic differentiation detection
MSC, EGFP-MSC and sTNFRⅡ-MSC were cultured in chondrogenic medium with or without TNF-α (20 ng/mL) for 14 days. Chondrogenic ability was assessed by alcian blue staining and western blot as described above. The dilution of primary antibody of aggrecan (AGG) (Novus) and SOX-9 (Novus) was 1:500.
Statistical analysis
Data in figures are shown as the means ± SD. ANOVA (SPSS Software Products) was used to analyze the data from multiple groups. A p values < 0.05 with a 95% confidence interval were considered significant.