Materials
Animals
BALB/cByJNarl, C57BL/6JNarl, B6.129-Tlr2tm1kir/J
Buffers
DPBS (Sigma), HBSS (Sigma), Golden lysis buffer [137 mM NaCl + 20 mM Tris-base + 5 mM EDTA + 1mM EGTA + 10% glycerol + 1% Triton X-100 + protease inhibitor cocktail (Roche) + phosphatase inhibitor cocktail (Roche)], ACK lysis buffer (150 mM NH4Cl + 10 mM KHCO3 + 0.1 mM Na2EDTA in distilled water)
Culture medium
Mouse MSC culture medium (MesenCultTM proliferation kit + 2 mM L-glutamine + 250 ng/mL amphotericin B), Adipogenesis kit (MesenCult), Osteogenesis kit (Gibco), Chondrogenesis kit (Gibco), Mouse CD4+ lymphocytes/total splenocytes and MSC coculture medium [RPMI 1640 medium (Hyclone) + 10% FBS (Gibco) + 2 mM L-glutamine (Biological Industries) + PSA (Biological Industries) + 15 mM pH7.0 HEPES (Sigma)]
Reagents
Polyinosinic-polycytidylic acid [poly (I:C)] (Sigma), Lipopolysaccharide [LPS]
(Sigma), Recombinant flagellin from S. typhimurium (Rec FLA-ST) (Invivogen), Recombinant TNF-a (PeproTech), Recombinant IFN-g (PeproTech), Pam3CSK4
(Invivogen), S3I-201 (Cayman), L-NMMA (Sigma), IL-6 neutralizing antibody (BD Pharmingen Cat. 554398), IgG1 k isotype control antibody (BD Pharmingen Cat.
554682), Anti-mouse CD16/32 (BD Pharmingen Cat. 553142), Foxp3/Transcription Factor Staining Buffer Set (eBioscience Cat. 005523), FITC anti-mouse CD25
(BioLegend Cat. 102005), FITC Rat anti-mouse CD34 (BD Pharmingen Cat. 560238), FITC anti-mouse CD80 (eBioscience Cat. 11-0801), FITC anti-mouse CD86 (BD Pharmingen Cat. 553691), FITC anti-mouse CD90.2 (eBioscience Cat. 11-0903), FITC anti-mouse Sca-1 (BioLegend Cat. 108105), PerCP-Cyanine5 anti-mouse CD4 (BD Pharmingen Cat. 550954), PE-Cyanine5 anti-human/mouse B220 (eBioscience Cat. 15-0452), APC anti-mouse CD11c (eBioscience Cat. 17-0114), APC anti-mouse CTLA-4 (BioLegend Cat. 106310), APC anti-mouse/rat Foxp3 (eBioscience Cat. 17-5773), PE anti-mouse/rat CD29 (BioLegend Cat. 102208), PE anti-mouse CD44 (BD Pharmingen Cat. 553134), PE anti-mouse CD73 (eBioscience Cat. 12-0731), PE anti-mouse CD105 (BioLegend Cat. 120408), Recommended isotype control antibodies for flow cytometry (BD Pharmingen or eBioscience), Mouse CD4+ T cell isolation kit (EasySep), Anti-CD3ε (BioLegend Cat. 100314), Anti-CD28 (BioLegend Cat. 102112), Mitomycin c (Sigma), 3H-thymidine (PerkinElmer), DNA polymerase kit (GoTaq), SYBR Green system (Bio-genesis), Ovalbumin (Sigma), Alum (Thermo Scientific), Anti-IgE, IgG1, IgG2a antibody for ELISA (BD Pharmingen), Antibodies for cytokine levels (R&D), TMB substrate (Clinical), b-actin antibody (Thermo Scientific Cat. MA5-15739), IkBa antibody (Cell Signaling Cat. 9247), NF-kB p65 antibody (Cell Signaling Cat.
8242), Phospho-NF-kB p65 (Ser 536) antibody (Cell Signaling Cat. 13346), STAT3 antibody (Flarebio Cat. CSB-PA004174), Phospho-STAT3 (Ser727) antibody (Cell Signaling Cat. 9134), NOS2 antibody (eBioscience Cat. 14-5920),
Acetyl-b-Pharmingen Cat. 558642), Anti-PE enrichment set (BD IMag), Type II collagenase (Worthington), Ficoll solution (GE).
Methods
Passage numbers 6 to 13 of characterized mouse BM-MSCs were used in this study. A TLR2 ligand, Pam3CSK4, was selected to generate the conditioned BM-MSCs.
[3H]-thymidine incorporation assays and the acute OVA-induced asthma murine model were used to examine the immunosuppressive activity of the conditioned BM-MSCs in vitro and in vivo, respectively. To investigate the signaling pathways of the Pam3CSK4
induction in BM-MSCs, STAT3 inhibitor, S3I-201 (Cayman), iNOS inhibitor, L-NG -monomethyl arginine citrate (L-NMMA) (Sigma), IL-6 neutralizing antibody (BD Pharmingen), or IgG1 k isotype control antibody (BD Pharmingen) was added in addition to the Pam3CSK4 treatment. On the other hand, to investigate the suppressive mechanisms of Mpam, the conditioned CD4+CD25+ T cells were generated by
coculturing with the conditioned BM-MSCs. [3H]-thymidine incorporation assays were further performed to examine the immunomodulatory effects of the conditioned
CD4+CD25+ T cells. The induction of CD4+CD25+Foxp3+ cells through conditioned BM-MSCs were elucidated in vitro and further investigated in vivo.
Additional details are described as follow:
Isolation and culture of MSCs from mouse bone marrow
BM cells were obtained from femurs and tibias of 4-5 weeks old female Balb/c mice, flushed with mouse MSC culture medium (MesenCult). MSCs were obtained by
expansion of the BM plastic-adherent cell fraction. Passage numbers 6 to 13 of BM-MSCs were used in this study.
Flow cytometry
Cells were processed according to the manufacturer’s protocols. Non-antigen-specific bindings of primary antibodies to the FcγII and FcγIII, and possibly FcγI receptors of cells were blocked by anti-mouse CD16/32 (BD Pharmingen Cat. 553142).
Non-hematopoietic surface markers CD29, stem cell antigen (Sca)-1, CD73, CD44, and CD105, and hematopoietic surface markers CD11b, B220, CD34, and CD11c were used to identify the non-hematopoietic MSCs. We performed intracellular staining using Foxp3/Transcription Factor Staining Buffer Set (eBioscience Cat. 005523). The flow cytometric acquisition was performed on a BD FACSCalibur device. The cells were acquired using forward scatter and side scatter and gated to exclude debris. The
cytometer then acquired 10,000 gated cell events. The fluorescence analysis was further conducted using BD CellQuestTM Pro software.
Differentiation assays
The cells were differentiated into adipocytes, osteocytes, or chondrocytes with lineage induction medium (MesenCult, Gibco, and Gibco, respectively). The
manufacturer’s instructions were followed to stain the differentiated cells. Adipocytes were detected by Oil Red O (Merck Millipore), osteocytes by Alizarin Red S (2 % Alizarin Red S in distilled water, adjusted to pH4.1-4.3 with NH4OH), and
chondrocytes by Alcian Blue (1 % Alcian Blue in 0.1 N HCl) staining.
Spleens from 5-8 weeks old female Balb/c mice were dissected. Red blood cells in total splenocytes were lysed using Ammonium-Chloride-Potassium (ACK) lysing buffer. CD4+ T cells in splenocytes were isolated by mouse CD4+ T cell isolation kit (EasySep) according to the manufacturer’s protocol. Total splenocytes or CD4+ T cells were respectively preactivated with soluble or coated 2 µg/mL anti-CD3ε (BioLegend) and 2 µg/mL anti-CD28 (BioLegend) for 24 h before being cocultured with mitomycin c (Sigma)-induced mitotically inactive BM-MSCs for 48 h. The cell ratio of total splenocytes or CD4+ T cells to BM-MSCs was 20 to 1. 1 µCi/mL 3H-thymidine
(PerkinElmer) was added into each well and the plate was incubated for 18 h. The cells were harvested and the thymidine incorporation was detected by a β-counter (MatrixTM 96). The same process was applied with the transwell insertions in the transwell assays.
In the transwell system, CD4+ T cells were seeded in the bottom well, while BM-MSCs were placed in the upper insertions.
Semi-quantitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR)
In vitro conditioned primary cells were collected after 24-h-conditioned treatment.
Total RNA was isolated from primary cultured cells and lung tissues using Trizol®
reagent. The RNA templates were reversed into first strand cDNA for PCR analysis (Fermentas). A DNA polymerase kit (GoTaq) and a SYBR Green system (Bio-genesis) were respectively used in semi-quantitative and real-time RT-PCR. In real-time PCR, gene expression levels were normalized to the housekeeping gene, gapdh, and
calculated using the 2^(-ΔΔCt) method. Please see appendix for the primer sequences used in this study.
Conditioned BM-MSCs
After being seeded for 24 h, BM-MSCs were either nontreated as the Mctrl or treated with a TLR2 ligand, Pam3CSK4 (InvivoGen) as the Mpam; a TLR3 ligand, polyinosinic-polycytidylic acid [poly (I:C)] (Sigma) as the M3; a TLR4 ligand, lipopolysaccharide (LPS) (Sigma) as the M4; a TLR5 ligand, recombinant flagellin from S. typhimurium (Rec FLA-ST) (Invivogen) as the M5; a recombinant TNF-a as the Ma (PeproTech); and a recombinant IFN-g as the Mg (PeproTech). To investigate the signaling pathway of immunosuppression-prone Mpam, a STAT3 inhibitor, S3I-201 (Cayman), an iNOS inhibitor, L-NMMA (Sigma), an IL-6 neutralizing antibody, or an IgG1 k isotype control antibody was added in addition to the Pam3CSK4 treatment to make Ms&p, Ml&p, M6&p, and Mic&p, respectively (Fig. 1A).
Acute OVA-induced asthma murine model
After acclimating to the Laboratory Animal Center for 1 week, 6-week-old female Balb/c mice were divided into four groups: the negative control (PBS), positive control (OVA), Mctrl treatment, and Mpam treatment groups. Each group originally had 9 mice in one experimental setting. Except for the negative control group, which was sensitized and challenged with PBS, they were intraperitoneally sensitized by 200 µl of 50 µg ova (Sigma)/2 mg Alum (Thermo Scientific) at day 0, 14, and 21, and then were intranasally challenged by 40 µl of 100 µg OVA at day 28, 29, and 30. After the third sensitization, OVA-sensitized mice with 0.5-0.7 ELISA units of serum OVA-specific IgE levels and negative control mice with less than 0.015 ELISA units of serum OVA-specific IgE levels were included in the following procedures. The OVA-sensitized groups were randomized into three groups based on their serum OVA-specific IgE levels. The
Mpam respectively, day 28 through day 30. The animals were either sacrificed or subjected to an airway resistance experiment on day 31 (Fig. 1B).
The composition of immune cells in lung
The bronchoalveolar lavage fluid (BALF) was collected from each sacrificed and tracheotomized mouse with 1 ml Hanks’ Balanced Salts (HBSS) buffer (Sigma). After the BALF was centrifuged at 1500 rpm for 5 min, the supernatant was used to examine the cytokine levels, while the nucleated cell pellet was resuspended with HBSS
containing 2% FBS to determine the cellular composition. The differential cell counts collected by Thermo ScientificTM CytospinTM 4 Cytocentrifuge were discriminated with Liu’s stain. The labels of the samples were covered by the other colleague during the counting processes, and a flow cytometric method was used to confirm the results (74).
Measurement of OVA-specific IgE levels in serum and OVA-specific cytokine levels in supernatants
The enzyme-linked immunosorbent assay (ELISA) protocol was followed to detect OVA-specific IgE (BD Pharmingen) and OVA-specific cytokine levels (R&D). The supernatants of the in vitro conditioned primary cells and the ex vivo restimulated splenocytes were collected after 72-h-conditioned treatment and 72-h-stimulation, respectively. The HRP-catalyzed oxidation of TMB substrate (Clinical) was finally conducted to detect the presence of the targets. And the optical density (O.D.)
differences between 540 nm and 450 nm wavelengths of the converted TMB were read on a standard electroimmunoassay plate reader (Molecular Devices, VersaMax).
Noninvasive measurement of airway hyperresponsiveness
The response of airway to aerosolized acetyl-b-methylcholine chloride (Sigma) in mice was detected using whole body plethysmography, and was analyzed using
FinePointTM system. The response of airway (Penh) to aerosolized acetyl-b-methylcholine chloride was then divided by the response of airway to aerosolized Dulbecco’s phosphate-buffered saline (DPBS) in each mouse, yielding Penh (%).
Invasive measurement of airway resistance
Airway function was measured by detecting changes in lung resistance (RL) in response to increasing doses of aerosolized acetyl-β-methylcholine chloride (Sigma) in anesthetized and tracheotomized mice. The calculation of RL was yielded by subtracting transpulmonary pressure (0.45 cmH2O.s.ml−1) from respiratory flow measurement. Data were presented as average RL in the ratio of average RL after PBS nebulization.
Histological evaluation
Mouse lung tissues were dissected and then fixed in a 10 % formaldehyde solution (Merck Millipore). Samples were embedded in paraffin blocks and the histological sections were stained by hematoxylin and eosin (H&E).
Conditioned CD4+CD25+ T cells
CD4+ T cells were prestimulated with coated 2 µg/ml CD3ε and 2 µg/ml anti-CD28 for 24 h before they were cocultured with mitomycin c-treated conditioned BM-MSCs in a 20:1 ratio. CD25+ cells were then isolated from these CD4+ T cells by anti-CD25 PE antibodies (BD Pharmingen Cat. 558642) and an anti-PE enrichment set (BD IMag) to make the Tctrl, Tpam, Ts&p, and Tl&p (Fig. 1C).
Griess assay
Mitotically inactive BM-MSCs were seeded as 1×105 cells overnight. The medium was then replaced by 250 µl of culture medium with treatments and incubated for 96 hours. The supernatants were assayed for nitric oxide by mixing 100 µl supernatant with 100 µl Griess reagent (Sigma) for 15 min at room temperature and were read on an electroimmunoassay plate reader at 550 nm. The concentration of nitrite in the culture supernatant was quantified by a standard curve of 100-0.003 µΜ NaNO2 concentrations (Sigma).
Western blot analysis
Protein samples were collected using Golden lysis buffer (137 mM NaCl, 20 mM Tris, 10 mM NaF, 5 mM EDTA, 1 mM EGTA, 10 % (v/v) glycerol, 1 % Triton X-100, protease and phosphatase inhibitor cocktails, pH 7.9), separated on 10 % SDS-PAGE gels, and transferred onto NC membranes (Whatman). After the membranes were incubated with primary and secondary antibodies, protein bands were visualized using chemiluminescence reagent (BIOSCIENCES). IκBα (Cell Signaling Cat. 4814), NF-κB p65 (Cell Signaling Cat. 3034), p- NF-κB p65 (ser536) (Abcam Cat. ab76302), STAT3 (Flarebio Cat. CSB-PA004174), p-STAT3 (ser727) (Cell Signaling Cat. 9134), iNOS (eBioscience Cat. 14-5920), and b-actin (ThermoFisher Cat. MA5-15739) were used as primary antibodies.
Pulmonary mononuclear cell preparation
The protocol established by D.L. Wiesner et al. was referenced (75). Lungs were excised and minced to generate pieces in HBSS (Sigma) + 1.3 mM EDTA (Sigma) solution for 30 min at 37°C with agitation. The solution was transferred to RPMI 1640
medium (Hyclone) supplemented with 10% FBS (Gibco) and 150 U/ml type II
collagenase (Worthington), and incubated for 1 h at 37°C with agitation. The cells were then grounded through a 70 µm filter, pelleted and resuspended with DPBS (Sigma).
Ficoll solution (GE) was finally used to separate mononuclear cells from the pulmonary homogenate.
Statistical analysis
Data were analyzed using GraphPad Prism v5 and are presented as mean and individual data points. To determine the significant variances between groups, Student’s t test was used between two groups, while one-way ANONA was used for multiple comparisons and Newman-Keuls Multiple Comparison Test was used for post hoc analysis. The statistical significance was set at p < 0.05.