Mouse Bone Marrow-Derived Mesenchymal Stem Cells (MSCs) and MH-S Cells
Mouse bone marrow-derived mesenchymal stem cells (MSCs) were purchased from Cyagen Biosciences (Guangzhou, China) and identified by cytometry (Fig.1A). The passage number of MSCs used in the experiment was under 15. MSCs were cultured in DMEM/F12 medium (Meilunbio, Dalian, China) with 8% fetal bovine serum (Cellmax, Beijing, China) and 1% penicillin/streptomycin (Meilunbio). MH-S cells were obtained from Otwo Biotech (Shenzhen, China) and cultured in DMEM medium (Gibco, Masschusets, USA) with 8% fetal bovine serum and 1% penicillin/streptomycin. All cells grew in a humidified 5% CO2 sterile incubator at 37°C.
In Vivo ALI Mouse Model
Male C57BL/6 mice, 8–10 weeks of age, were purchased from Zhejiang Academy of Medical Sciences (Hangzhou, China). The mice were kept in Sir Run Run Shaw Hospital (Hangzhou, China) with a 12-h light/dark cycle and free access to food and water. The mice were anesthetized and instilled with Lipopolysaccharide (LPS, 5 mg/kg for most experiments or 10 mg/kg for the survival experiment) (Escherichia coli, 0111:B4; Sigma-Aldrich, USA) in 50 ml of PBS through a 24G catheter inserted into the trachea, establishing the ALI model. Control mice were instilled with 50 ml of PBS in the same way. All experiments were performed in conformity to Institutional Animal Care and Use Committee-approved protocols.
The experimental protocol is shown in Fig. 1B. The mice were divided into three groups: PBS+PBS group, LPS+PBS group, and LPS+MSC group. MSCs (2×106 cells per mouse) in 50 ml of PBS or PBS alone were instilled intratracheally (IT) at 4 h after LPS instillation in the LPS+MSC group and the other groups, respectively. The mice were euthanized 48 h after ALI model establishment for further experiments or observed for 2 weeks to record their body weight and survival rate
Mouse in Vivo imaging
Luc-PGK-Puro lentivirus was transfected to MSCs following the manufacture’s instructions, the stable luciferase expression MSCs were obtained after puromycin filtration (named Luc+MSC). The mice were divided into PBS instilled group (control group) and LPS instilled group (ALI model group). Luc+ MSC (2×106 cells per mouse) in 50 ml PBS was instilled intratracheally (IT) at 4 h after model establishment. D-luciferin(15mg/ml) was injected intraperitoneally (10ul/g) at 2h, 24h and 48h after Luc+ MSC instillation. Fluorescence signal was detected at 30 min after D-luciferin injection with imaging system (PerkinElmer, Caliper IVIS Lumina II, USA).
Arterial Oxygenation Analysis
For arterial oxygenation analysis, mice were first anesthetized and the carotid artery was adequately exposed. The distal end was ligatured with silk while the proximal end was occluded. A PE-10 catheter was then inserted through a V notch and arterial blood was collected using a 1 ml heparinized syringe through the catheter after removing the occlusion. The mice were then euthanized for other experiments. A blood gas analyzer was used to determine arterial oxygenation.
Bronchoalveolar Lavage Fluid
For the collection of bronchoalveolar lavage fluid (BALF), mice were euthanized, after which the trachea was exposed quickly and then cannulated using a 24G catheter. This catheter was fixed using silk ligature. A total of 0.8 ml of cold PBS-EDTA solution (2 mM) was infused, which was maintained for 1 min to collect BALF. This was repeated five times and the collected BALF was temporarily stored on ice.
Pulmonary Capillary Permeability Measurement
For measurement of the permeability of pulmonary capillaries, the right upper lobe of the lungs was removed and its wet weight was recorded. Then, its dry weight was measured after placing the lungs in a 70°C incubator for 24 h. The wet/dry weight ratio was then calculated. The collected BALF was filtrated with a 40 μm strainer and then centrifuged at 300g for 5 min. The resulting supernatant was collected, the protein content of which was measured by BCA assay.
For histological analysis, the right lobe of the lungs was excised, fixed with 10% formalin for 24 h, embedded in paraffin, cut into 5 μm slices, and finally stained with hematoxylin and eosin.
For flow cytometry, the centrifuged cells in BALF were suspended in PBS, incubated for 10 min with FcR blocking reagents, and then incubated for 15 min with APC-CY7 fixable viability dye, followed by another 20 min with PE-F4/80, FITC-CD11b, and PE-CY7-CD86 antibodies (Biolegend, USA). After incubation, the cells were washed with PBS twice, after which R-PE IMag particles (BD Pharmingen, USA) were added and incubated for 30 min on ice, followed by enrichment of the PE-labeled cells by magnetic cell sorting with a magnetic frame. The remaining cells were then washed twice and fix/perm treatment was performed, after which the cells were incubated with APC-CD206 (Biolegend, USA) antibody for 20 min. The stained cells were washed and suspended in 300 ml of buffer for flow cytometry (BD LSRFortessa, USA). The cultured MH-S cells were incubated with FcR blocking reagents, APC-CY7 fixable viability dye, FITC-CD11b, and PE-CY7-CD86 antibodies for flow cytometry. The results were analyzed by FlowJo software.
Co-culture of MSCs and MH-S Cells
For the co-culture of MSCs and MH-S cells, a six-well transwell co-culture system (0.4 μm pores; Corning, USA) was applied. MSCs (2×105 cells/well) were seeded in the six-well plates and MH-S cells (5×105 cells/well) were grown in the upper wells of the six-well transwell co-culture system overnight. Then, the upper wells were transferred to the lower wells that contained MSCs, establishing a co-culture system, and the medium was changed to DMEM/F12 complete medium with LPS (200 ng/ml) and IFN-γ (20 ng/ml), which were used for activating MH-S cells. NS-398 (10 μM; APExBIO, USA) or GW627368X (10 μM; MCE MedChemExpress, China) was added to the medium as needed. The cells were harvested after 24 h for subsequent experiments.
For western blotting, the cells were lysed in cell lysis buffer (Beyotime, China) with protease inhibitor. Proteins were separated by SDS-PAGE and transferred to PVDF membranes, after which the membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature. These membranes were then incubated with the primary antibodies overnight at 4°C on a rotator. Membranes were then incubated with HRP-conjugated secondary antibody (Beyotime) at room temperature for 2 h, enhanced chemiluminescence reagents were added to visual bands, and the grayscale value was quantified using ImageJ software. The primary antibodies used were for iNOS, GAPDH (CST, USA), and COX2 (Bioworld, China).
Quantitative Real-time PCR (qPCR)
For quantitative real-time PCR, total RNA was isolated from cells using a Total RNA Isolation Kit (Vazyme, China) and measured for optical density at 260 nm. Subsequently, total RNA was reverse-transcribed to cDNA with a reverse transcription kit (Vazyme). qPCR was carried out with a two-stage program. Primers used for qPCR were for mouse TNF-α, Ccl2, Ccl3, Ccl5, Cxcl2, and GAPDH (Tsingke Biotechnology, China). The sequences were as follows: mouse TNF-α: 5’-CCCTCACACTCAGATCATCTTCT-3’(F), 5’-GCTACGACGTGGGCTACAG-3’(R), Ccl2: 5’-TTAAAAACCTGGATCGGAACCAA-3’(F), 5’-GCATTAGCTTCAGATTTACGGGT-3’(R), Ccl3: 5’-TTCTCTGTACCATGACACTCTGC-3’(F), 5’-CGTGGAATCTTCCGGCTGTAG-3’(R), Ccl5: 5’-GCTGCTTTGCCTACCTCTCC-3’(F), 5’-TCGAGTGACAAACACGACTGC-3’(R), Cxcl2:5’- CCAACCACCAGGCTACAGG-3’(F), 5’-GCGTCACACTCAAGCTCTG-3’(R), and GAPDH: 5’-AGGTCGGTGTGAACGGATTTG-3’(F), 5’-TGTAGACCATGTAGTTGAGGTCA-3’(R).
Enzyme-Linked Immunosorbent Assay (ELISA)
The concentrations of TNF-α, Ccl2 and PGE2 factors were measured using ELISA with mouse TNF-α and Ccl2 ELISA kits (Elabscience, China) and PGE2 ELISA kits (R&D, USA), following the manufacturers’ instructions.
Bulk RNA-seq and Data Analysis
Rest MSCs before co-culture and activated MSCs after co-culture were collected (Fig. 5A), after which their total RNA was extracted, quantified, and qualified. RNA sequencing was performed with an Illumina platform. Raw reads were first processed using in-house Perl scripts. Then, clean reads were obtained by removing reads containing an adapter, reads containing a poly(N) region, and low-quality reads from the raw reads. A reference genome and gene model annotation files were directly downloaded from a genome website. The reference genome index was built and paired-end clean reads were aligned to the reference genome by Hisat2 v2.0.5. Then, featureCounts v1.5.0-p3 was applied to count the reads mapped to each gene. Analysis of differentially expressed genes (DEGs) between two groups was performed using the DESeq2 R package (1.16.1). Genes with an adjusted p-value <0.05 found by DESeq2 were considered to be differentially expressed. A cluster heatmap of the DEGs was created using the ComplexHeatmap R package (2.6.2). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed with the web-based Gene SeT AnaLysis Toolkit (http://www.webgestalt.org). To construct a transcription factor (TF)–target gene (TG) network, we collected mouse TF-TG interactions from TRED and KEGG databases, resulting in a list of TF-TG pairs (Supplemental S1). Screening was performed to identify the target genes and transcription factors among the DEGs and the network was visualized using Cytoscape software.
All statistical analyses were performed using SPSS 23.0 software and the data were visualized using GraphPad Prism 7.0. Kaplan-Meier test was applied for survival curve comparison. Comparisons between multiple groups were performed using one-way analysis of variance (ANOVA). Comparisons between two groups were performed using T test. The data are expressed as the mean ± SD and a value of p<0.05 was considered significant.