Stem Cell

Inhibition of lipopolysaccharide-induced inflammation by trophoblast-conditioned medium and trophoblast-derived extracellular vesicles in human middle ear epithelial cells – Scientific Reports

Time Stamp: November 13, 2023 7:00 PM
Source Node: 269900

Cell culture

HMEEC (cells into which human papillomavirus E6/E7 genes were inserted), were provided by Dr. David at the House Ear Institute in the USA. Cells were cultured in an airway epithelial cell growth medium (C-21160; PromoCell, Heidelberg, Germany) and supplement (C-39165; PromoCell, Heidelberg, Germany). HTR-8/SVneo was purchased from ATCC (CRL-3271; VA, USA) and cultured in RPMI-1640 (11875-093; Gibco, Paisley, UK) with 1% penicillin/streptomycin (P/S) (15140-122; Gibco, Grand Island, USA) and 5% fetal bovine serum (FBS) (16000-044; Gibco, Grand Island, USA). All cells were cultured in humidified air in a 37 °C incubator, containing 5% CO₂.

Preparation of TB-CM and TB-EVs

To obtain TB-CM, HTR-8/SVneo cells were rinsed twice with phosphate-buffered saline (PBS) (10010; Gibco, NY, USA) upon 60% confluence. The medium was replaced with RPMI-1640 medium (Gibco) with 5% exosome-depleted FBS (Gibco) and 1% P/S (Gibco). CM was incubated for 48 h and collected in a 50 mL tube. CM was obtained by centrifugation (Union 32R Plus; Hanil Science, Inchon, Korea) at 300×g for 10 min to isolate cellular waste, followed by filtration with a syringe filter (pore size 0.22 µm) (CLS431229; Corning, NY, Germany). To isolate TB-EVs from the supernatant following the removal of cellular debris in TB-CM, we used an ultracentrifuge (Optimal Max-Xp, Beckman Coulter, CA, USA). The supernatant was first centrifuged at 300×g for 10 min at 4 °C, and the upper layer was collected. Subsequently, another centrifugation was performed at 3000×g for 10 min at 4 °C, and the upper layer was collected. Finally, the pellet was obtained by centrifuging at 100,000×g for 150 min at 4 °C and then diluted in filtered PBS for recovery.

Cell counting Kit-8 assay (CCK-8 assay)

HMEEC viability was assessed using a CCK-8 assay (CK04; Dojindo, Kumamoto, Japan). HMEECs were inoculated at a density of 1 × 10⁴ cells per well in a 96-well dish and incubated for 24 h. Cells were simultaneously treated with LPS (5.6 µg/mL; Sigma, St. Louis, MO, USA) and TB-CM (0, 10, 15, 20, 25, and 30%), with a control group without LPS and TB-CM addition. After incubation at 37 °C for 24 h or 48 h, a total volume of 10% CCK-8 solution was added to each well. The cells were then incubated at 37 °C for 1 h. Subsequently, the optical density was gauged at a wavelength of 450 nm with the aid of a microplate reader (SpectraMax 190 Microplate Reader, California, USA).

BrdU assay

Cell proliferation was assessed using BrdU immunocytochemistry. HMEECs were cultured in 24-well plates with 25 µM BrdU (B5002; Sigma, St. Louis, USA), TB-CM, TB-EVs, and LPS. After 24 h, the cells were rinsed with PBS and subsequently immobilized with 4% paraformaldehyde (PFA) at 4 °C for 20 min. For DNA denaturation, the cells were incubated with 1N HCl at 4 °C for 10 min, 2N HCl at 17 °C for 10 min, and 2N HCl at 37 °C for 20 min. The cells were cultured with 0.1% Triton X-100 and 3% BSA in PBS at 37 °C for an hour. BrdU monoclonal antibody Alexa fluor 488 (1:1000, B35130; Invitrogen) was incubated at 4 °C for 24 h. After washing, it was observed with an automated fluorescence microscope (Evos FL Auto 2; Invitrogen, WA, USA).

Quantitative real-time PCR

TRIzol™ reagent (15596018; Invitrogen, CA, USA) was applied to extract total RNA from every set of HMEECs. Add 1 ml of TRIzol™ reagent to each group to lyse the cells. Add 200ul of chloroform (C2432-500ML, Sigma, USA), mix well, incubate for 3 min, and then centrifuge at 19,000×g for 20 min. Collect only the supernatant, add 2-propanol (19516-500ML, Sigma, Germany) in a 1:1 ratio, and gently invert. Centrifuge at 19,000×g for 20 min. Completely remove the supernatant, perform two washes with 70% ethanol, discard the supernatant. Dissolve the pellet in sterilized distilled water, measure the RNA concentration using a nanodrop (AZY1707715, Thermo Scientific, USA). To synthesize cDNA, 1 μg of RNA was subjected to reverse transcription using a PrimeScript™ 1st strand cDNA synthesis kit (6210A; Takara, Kusatsu, Shiga). The RT-qPCR mixture constituted 1 µl cDNA, 5 pmol of forward and reverse primers, and 10 µl of SYBR green Master Mix (Life Technology, Warrington, UK), for a total volume of 20 µl. RT-qPCR was completed using the Quant Studio 6 Flex system. The denaturation process was performed at 95 °C for a duration of 15 s, followed by annealing at 60 °C for a period of 1 min, and 40 cycles were repeated. All experiments were run in triplicate. The Ct value was calculated using a relative quantification method by normalizing the mRNA expression levels of GAPDH. The primer sequences data used for RT-qPCR are listed in Tables S1 and S2.

Western blot analysis

Thermo Scientific’s NE-PER™ reagents (78,833; Thermo, Rockford, USA) were used to extract nuclear and cytoplasmic proteins from HMEECs. The PER™ reagents was used according to the manual⁵. The total protein was incubated for 1 h at 4 °C in RIPA buffer. Cells were lysed and quantified with the use of a bicinchoninic acid (BCA) assay to achieve the same quantities of protein. TNF-α (1:1000, SC-52746; Santa Cruz), COX-2 (1:1000, ab52237; Abcam), ALIX (1:1000, EXOAB-ALIX-1; SBI), CD63 (1:1000, EXOAB-CD63-1; SBI), CD81 (1:1000, EXOAB-CD81A-1; SBI), COMMD5 (1:1000, 10393-1-AP; Proteintech), NF-κB (1:1000, SC-8008; Santa Cruz), Phospo-NF-κB (1:1000, SC-136548; Santa Cruz), and β-actin (1:1000, 8H10D10; Cell Signaling) were used as antibodies and incubated for a day at 4 °C. Anti-Mouse IgG-HRP (1:4000, 7076S; Cell signaling) and anti-Rabbit IgG-HRP (1:4000, 7074S; Cell Signaling) was added for 2 h at room temperature. Bands were visualized using chemiluminescence (SuperSignal™ West Femto Maximum Sensitivity substrate; Meridian Rd., Rockford, USA). Chemiluminescence analyzer system was used to visualize the bands, which was done within 10 min.

Immunofluorescence staining

HMEECs were seeded at 2 × 10⁵ cells/well on a cell culture slide (8 wells; SPL Life Sciences) and incubated overnight. The cells were rinsed twice with PBS and induced with LPS (56 µg/mL) and TB-CM (10% and 20%) in the growth medium. Controls were not treated with LPS or TB-CM. Cells were cultured for 24 h, washed twice with PBS, and fixed with 4% PFA for 15 min. The cells were then incubated in 0.3% Triton X-100 (in PBS) as a permeabilization solution at 37 °C for 5 min. BSA (3% in PBS) was added to the permeation solution and incubated for blocking for 1 h at 37 °C. The primary antibody, MUC5AC (1:500, MA1-38,223; Thermo Fisher) was diluted in blocking buffer and incubated at 4 °C for 24 h. After washing thrice for 5 min, Alexa Fluor™ 488 goat anti-mouse IgG (H + L) (1:2000, A11001; Invitrogen) was incubated for 2 h at 37 °C in the dark. They were washed thrice for 5 min with PBS, and stained nuclei with DAPI. Cellular fluorescence signals were observed with a confocal microscope (Zeiss LSM700, Oberkochen, Germany) and images were analyzed with ZEN lite (version 2.3.)

RNA-sequencing

To identify DEG, we conducted RNA sequencing. In the library preparation phase, we isolated total RNA from LPS-induced HMEECs and LPS-induced HMEECs treated with TB-CM (20%) for 24 h. We constructed transcriptome libraries following the manufacturer’s protocol using the TruSeq Stranded mRNA Reference Guide (Illumina, San Diego, CA, USA)⁴⁶. We generated cDNA from fragmented RNA through reverse transcription, attaching different adapters to both ends of the cDNA fragments and ligating them. After PCR amplification to obtain an insert size of 200–400 bp, we performed size selection to ensure the quality of the reads obtained through sequencing. We conducted an overall read quality analysis, including total bases, total reads, and GC (%) statistics. To reduce bias in the analysis results, we underwent a preprocessing step to remove artifacts such as low-quality sequences, adapter sequences, contaminant DNA, and PCR duplicates. Preprocessed reads were then mapped to the reference genome using the HISAT2 program (v2.1.0). Using information from the reference-based aligned reads, we extracted expression profiles in terms of FPKM (Fragments Per Kilobase of transcript per Million mapped reads)/RPKM (Reads per Kilobase of transcript per Million mapped reads) values and TPM (Transcripts Per Kilobase Million) values, considering transcript length and depth of coverage⁴⁷. We selected genes or transcripts with statistically significant differential expression values among more than two groups with different conditions using statistical hypothesis testing. To identify genes differentially expressed between the two groups, we extracted genes that satisfied the condition |fc|> = 2 and exactTest raw p-value < 0.05 using edgeR for the comparison groups (LPS and LPS + TB-CM). For DEGs with known gene information, we performed functional annotation and gene set enrichment analysis based on GO and KEGG pathways (https://www.genome.jp/kegg/pathway.html). GO analysis was conducted from three perspectives using the DAVID website (https://david.ncifcrf.gov/tools.jsp). To examine gene networks, we utilized Cytoscape (v3.9.1) tools and the stringAPP for analysis.

siRNA transfection

siRNA sequences were designed for COMMD5 (5′-GUU UUA GGU UGG GCA UUU U-3′, 5′-AAA AUG CCC AAC CUA AAA C-3′) (Bioneer, Daejeon, South Korea). HMEECs were cultured in 12-well plates for one day at a cell confluency of 60–70%. Four hours before transfection, plates were washed twice with PBS and replaced with basal culture medium without added P/S and FBS. The transfected cocktail was prepared with 400 pmol of siRNA and 8 µl of lipofectamine in DMEM/F12 medium using Lipofectamine RNAiMAX (Invitrogen, Van Allen Way Carlsbad, CA, USA). Cells were then incubated for 15 min at room temperature to mix the complexes and 200 μl of transfection cocktail was added to each well. After 24 h, cells were harvested for Western blot assay and RT-qPCR analysis.

NTA

EV size, concentration, and distribution were confirmed using NTA. For particle light scattering recordings, we utilized NTA equipment (Malvern, NS300, Grove Wood Road, UK), with a scientific CMOS (sCMOS) camera type and a red laser type. The samples were prepared by diluting TB-EVs (1 mL) at a ratio of 1:20. The laser irradiated the sample, while the camera captured the movement of particles. Three videos, recorded for 60 s each, were analyzed to visualize EV concentration, size, and distribution.

Flow cytometry

To characterize microvesicles, microvesicles isolated from TB-CM were used for flow cytometry. Since exosomes are too small to be directly used for flow cytometry, exosomes were captured and analyzed with a bead with a diameter of 9.1 μm. Exosome cell surface protein markers CD63 (Exo-Flow Capture kit, EXOFLOW300A-1; SBI) were prepared according to the manufacturer’s instructions. Next, Exo-Flow FACS magnetic beads were prepared, and the bead-attached capture antibody was incubated at 4 °C for 24 h. Exosomes were then stained using Exo-FITC exosome satin, and cytometric data were measured using a Flow Cytometer Analyzer (BD LSRFortessa™ X-20 special order system, CA, USA).

Statistical analysis

All statistics outcomes are expressed as averages of three or more experiments. To determine the importance among groups, the statistical evaluation software program PRISM 5.1 (San Diego, CA, USA) was used, and a two-way analysis of variance was performed. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.