Study protocol
Differentiated human intestinal cell line Caco-2 (ATCC HTB37) was exposed to either 5 % CO2 and 95 % air (normoxia group) or 5 % O2, 5 % CO2, and90 % N2 (hypoxia group) for 6 h. After 6 h, the culture medium was refreshed. Each group was subsequently incubated for 24 h at 5 % CO2 and 95 % air. The medium of each group was collected and filtered at the end of the incubation, and the exosomes were extracted.
Subsequently, monocyte NF-κB reporter assay, proteome analysis using high performance liquid chromatography–mass spectrometry (HPLC-MS), and miRNA quantification were conducted using real-time polymerase chain reaction (RT-PCR; Fig. 1).
Cell culture
Differentiated Caco-2 and THP1-Blue NF-κB cells (InvivoGen, San Diego, CA, USA) were used in the experiment. Caco-2 is a carcinoma epithelial cell line of colonic origin with small intestinal enterocyte-like features.26 They were cultured in Dulbecco’s modified Eagle medium (Nakaraitesk, Kyoto, Japan) supplemented with 2 mM L-glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 % fetal bovine serum (FBS). After 3 days of culture, cells were harvested by trypsinization. THP-1 is a monocyte cell line engineered to monitor NF-κB activation by determining the activity of secreted embryonic alkaline phosphatase (SEAP). Roswell Park Memorial Institute 1640 medium, supplemented with 2 mM glutamax, 10 μg/mL blasticidin, 50 μg/mL streptomycin, 50 U/mL penicillin, 100 μg/mL normocin, 1 mM sodium pyruvate, and 10 % endotoxin-free heat-inactivated FBS, was used for the selection pressure. The cells were maintained in 75 cm2 flasks at 37 °C in a humidified incubator containing 5 % CO2. The medium was changed daily until confluency was achieved and every other day thereafter.
Isolation of exosomes
The supernatant was centrifuged to remove cells and cellular debris at 3,000 × g for 15 min. The particle suspension was then centrifuged using the Amicon Ultra-15 (Sigma-Aldrich, St. Louis, MO, USA) Centrifugal Filter Unit with 100 kDa cutoff at 4,000 × g for 20 min at 25 °C for concentration. Subsequently, ExoQuick-TC Exosome Precipitation Solution Kit (System Biosciences, Palo Alto, CA, USA) was used for exosomal extraction following the manufacturer’s instructions: the samples (250 μL) were centrifuged at 3,000 ×g for 15 min to remove cells and cellular debris. The samples were then transferred to a sterile vessel, 63 μL ExoQuick Exosome Precipitation Solution was added, and the samples were incubated overnight. The next day, the samples were centrifuged at 1,500 ×g for 30 min, and the exosomes in the pellet were obtained by aspiration of the supernatant.
Immunoblotting of exosomal surface proteins
Isolated exosomes wereverifiedvia immunoblotting with Exo-CheckAntibody Array(System Biosciences) containingpre-printed spots ofeightantibodiesagainstknown exosome markers (FLOT1, ICAM, ALIX, CD81, CD63, EpCAM, TSG101, and ANXA5). The experimental conditions were carried out in accordance with the manufacturer’s instructions.
Transmission electron microscope
Exosomal specimen preparation for use with a TEM was performed by the personnel of the Instrumental Analysis Division at the Research Center of Tokyo Medical and Dental University. Briefly, the exosomes were immersed for 4 h (Karnovsky’s fixative: pH 7.42, 2.5 % glutaraldehyde and 2% paraformaldehyde in a 0.15 M sodium cacodylate buffer), post-fixed for 60 min (0.15 M cacodylate buffer and 1 % osmium tetroxide), and stained in 2 % uranyl acetate for 60 min. Samples were treated with pure ethanol for dehydration, resin-embedded, and sectioned at 50–60 nm using Ultramicrotome Leica EM UC7 (Leica Microsystems, Wetzlar, Germany). Staining was performed using 2 % uranyl acetate for 5 min and Sato’s lead stain for 1 min; 27 grids were viewed using a H7100 Transmission Electron Microscope (Hitachi High-Technologies, Tokyo, Japan), equipped with an XR81 (8 megapixel) charge-coupled device camera (AMT Imaging Systems, Woburn, MA, USA).
Nanoparticle tracking analysis
A NanoSight LM10 (Malvern Instruments, Malvern, UK) supplied with a fast video capture and NTA software version 2.3(Malvern Instruments, Malvern, UK) were used for particle detection, counting, and sizing. The experiment was performed as per the manufacturer’s instructions. Calibration was performed using 100 nm polystyrene beads (Thermo Fisher Scientific, Fremont, CA, USA) prior to measurement. The exosomal fraction was diluted at 1:256 using sterile phosphate-buffered saline. Briefly, recordings were taken every 60 s and repeated thrice for each sample. The size distribution of the particles was expressed in nm.
Monocyte NF-κB reporter assay
A THP-1 Blue NF-κB reporter monocyte cell with a concentration of approximately 5 × 105 cells/mL was added to each well of a 96-well flat-bottomed plate, which was then incubated with 20 μL of each sample at 37 °C for 18 h. After incubation, 20 μL of each well were extracted and mixed with 180 μL of QUANTI-Blue (InvivoGen) in a new 96-well plate. The plate was incubated at 37 °C for 4 h, and the SEAP levels were quantified using a spectrophotometric microplate reader (VersaMax Microplate Reader, Tokyo, Japan) at 620 nm and SoftMax Pro 5.3 software (Molecular Devices, CA, USA).
Lipid extraction
Lipids in the exosomes were extracted using the Bligh and Dyer technique.27 The chloroform-methanol-aqueous (1:2:0.8, v/v/v) mixture was stirred at 4 °C for 30 min. Then, 10 mmol/L of Tris-HCl (pH 7.4) and chloroform were added. The mixtures were centrifuged for 15 min at 1000 × g. For internal standards, 1,2-dilauroyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Alabaster, AL, USA) and 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine and were added. The upper and the lower phases were collected using chloroform and nitrogen gas stream respectively. Lipids obtained by the above process were dissolved with chloroform-methanol (2:1, v/v) and stored at -20 °C.
HPLC/ESI-MS/MS-based lipidomic analysis
For lipidomic analyses, HPLC/ESI(electrospray ionization)-MS/MS was performed. A Dionex Ultimate 3000 UHPLC system (Thermo Fisher) was coupled to a QTRAP5500 triple quadrupole-linear ion trap mass spectrometer (AB SCIEX, Foster, CA, USA). The Develosil C30-UG-3 column (Nomura Chemical, Tokyo, Japan) were used for LC separations. The flow rate was 80 μL/min.Mobile phases consisted of acetonitrile-methanol-water(2:2:1, v/v/v), containing 1 mM ammonium formate and 5 µM phosphoric acid(A), and 2-propanol containing 1 mM ammonium formate and 5 µM phosphoric acid acetonitrile (v/v) (B). The gradient profile was as follows: 0–5 min, 0 % B; 5–60 min, 80 % B; and 60–70 min, 0 % B for re-equilibration. The analyses were performed in negative ion mode, using multiple reaction monitoring approaches. The software AB SCIEX (AB SCIEX, Foster, CA, USA) was used for analyzing LC chromatogram peaks.
Shotgun protein analysis
For protein analysis, a combination of Nano-HPLC Chromatography System and timsTOF Pro (Bruker Daltonics Billerica, MA, USA) was used. To ensure reproducibility, the experiments were carried out on identical two samples (The samples placed in hypoxic conditions are labeled H1 and H2, and the samples placed in normoxic conditions are labeled N1 and N2) The mass spectrometer was operated in parallel accumulation serial fragmentation (PASEF) mode. Samples were firstly desaltated and concentrated using C18 Omix (Agilent Technologies, Santa Clara, CA, USA). NanoElute ultra-high pressure nanoflow chromatography system (Bruker Daltonics) was used for the chromatography step. Proteins were concentrated onto an Acclaim PepMap 100 C18 precolumn (5 mm × 300 μm i.d.; Thermo Fisher Scientific) and subsequently separated onto an Aurora reversed phase ReproSil column (25 cm × 75 μm i.d.) at 50 °C. Mobile phases consisted of 0.1 % formic acid, 99.9 % water (v/v) (A), and 0.1 % formic acid in 99.9 % acetonitrile (v/v) (B). The nanoflow rate was 400 nL/min, and the gradient profile was as follows: 0–60 min, 2 % B; 60–90 min, 17 % B; 90–100 min, 25 % B, 100–110 min, 37 % B, 110–120 min 95 % B, followed by a washing step at 95 % B and re-equilibration.
TimsTOF Pro mass spectrometer (Bruker Daltonics) with a modified nano electrospray ion source (CaptiveSpray, Bruker Daltonics) was used for the MS experiments. Spray voltage was 1600 kV, and the capillary temperature was set at 180 °C for ionization. MS spectra were acquired in the positive mode in the mass range from 100 to 1700 m/z.
The outlet of the reverse phase nanocolumn was connected to the Agilent 1100 Series LC/MSD Ion Trap SL. The eluting peptides were analyzed by data-dependent tandem MS to obtain mass and sequence information for further database analysis. The identification of the proteins was conducted using the Mascot software program (Matrix Science, Chicago, IL, USA; version 1.7).27
MiRNA analysis
Total RNA was isolated from cell lines using the miRNeasy Mini Kit (Qiagen, Duesseldorf, Germany).The expression levels of each miRNA were examined using the RT2 miRNA PCR Arrays Human miFinder kit(Qiagen). RT2 Easy First Strand Kit (Qiagen) was used to synthesize cDNA from each sample. Subsequently, 100 μL of cDNA template, 1,275 μL of 2X RT2 SYBR Green Fluor qPCR Master Mix (Qiagen), and double-distilled H2O were added to obtain a final volume of 2,550 μL. The mixture of an equal volume was dispensed into a 96-well plate. A 25 μL reaction system was prepared, and the Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used for the PCR assay. The PCR cycling program was pre-incubated to 95 °C, and then 40 cycles of the following were completed: denaturation, 15 s at 94 °C; annealing, 30 s at 55 °C; and extension, 30 s at 70 °C. The raw data were exported using the Applied Biosystems 7500 Sequence Detection System. Differentially expressed miRNAs were analyzed using Qiagen’s online analysis software.
Statistical analysis
The Student's t-test was performed to test the differences between the samples with and without ischemic stimulation. This study includes a comprehensive omics analysis and does not assume a null hypothesis of an increase or decrease in any substance. For this reason, no significance level was set, and no sample size determination was made.Volcano plots29 were also generated for miRNA and protein results. All the statistical analyses were performed using R software (version 3.6.0; R Foundation for Statistical Computing, Vienna, Austria).
Bioinformatic analysis
For the interpretation of the obtained results, bioinformatics approach was adopted. Proteins were analyzed with gene ontology (GO) term enrichment analysis using g:Profiler (https://biit.cs.ut.ee/gprofiler/gost), functional enrichment analysis and protein-protein interaction network functional enrichment analysis was performed using STRING database (https://string-db.org/).