Patients and exosome isolation
The study was approved by the Institutional Review Board at Hallym University Kangnam Sacred Heart Hospital (IRB no. 2022-03-020). Exosomes were isolated from blood samples from healthy controls and psoriasis patients using an exosome purification kit (Cells guidance systems, St. Louis, MO, USA). Briefly, blood samples were centrifuged at 1,500 rpm for 15 min to collect the supernatant, which was further centrifuged at 300 × g for 10 min. The resulting supernatant was centrifuged at 16,000 × g for 30 min and again at 16,000 × g for 60 min. The pellet containing exosomes was resuspended in 200 µl of PBS in a 1.5 mL tube, followed by centrifugation at 50 g for 60 sec to obtain the exosomes.
ADSC isolation and culture
Human adipose tissue was obtained through liposuction surgery with informed consent from a 30-year-old male. Adipose tissue was washed with a phosphate buffer solution to remove red blood cells, followed by incubation with 0.075% collagenase-II (Sigma‒Aldrich, St. Louis, MO, USA) at 37°C for 40 minutes. After centrifugation and filtration through a 100 µm cell strainer, the resulting cells were cultured in expansion media containing α-MEM (Welgene, Gyeongsan, Gyeongsang, Korea), 10% FBS (Capricorn Scientific GmbH, Ebsdorfergrund, NRW, Germany), and 5 ng/ml recombinant bFGF (R&D system, Minneapolis, MN, USA). The expansion medium was replaced every 2 days, and the cells were subcultured until passage 6.
Preparation of ADSC-conditioned media
ADSCs were seeded at 3.7x109 cells/cm2 and cultured in the expansion media for 3 days. Then, to remove FBS and particles, the cells were cultured in α-MEM without FBS. After 24 h, the cells were washed with phosphate buffer solution and cultured in α-MEM containing 10% exosome-depleted SR (XenoFree CTS; Gibco Life Technologies, Carlsbad, CA, USA) for 2 days. The ADSC-conditioned media were harvested and filtered through a 0.22-µm filter.
Separation of ADSC-derived exosomes via a TFF system
Exosomes from ADSC-conditioned media were isolated using a TFF system equipped with a 500-kDa cutoff hollow fiber membrane (EMD Millipore Corp., Billerica, MA, USA). The conditioned media (1 L) was processed through the TFF system, maintaining a fluid velocity of 180 r/min and a pressure below 1.5 psi. Subsequently, PBS (2 L) was introduced into the TFF system to enhance exosome purity and buffer exchange. The resulting exosomes were then filtered through a 0.22-µm filter.
The size and quantity of exosomes were determined using nanoparticle tracking analysis (NTA) (NS300; Malvern Instruments, Malvern, Worcestershire, UK). The protein content of the exosome solution was measured using the BCA assay (iNtRON biotechnology, Seongnam-si, Gyeonggi-do, Korea). Exosomes were stored at -80°C for future use.
Cell Culture and treatments
The immortalized human keratinocyte cell line HaCaT (Welgene, Daegu, Korea) was cultured in Dulbecco's modified Eagle's medium (DMEM) (Lonza, Walkersville, MD, USA). DMEM was supplemented with 1% penicillin‒streptomycin and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and incubated in a CO2 incubator at 37°C (5% CO2). HaCaT cells were treated with healthy control serum-derived exosomes (2.5 x 108/㎖), psoriasis serum-derived exosomes (2.5 x 108/㎖), ADSC-derived exosomes (3.7x109/㎖), healthy control serum-derived exosomes (2.5 x 108/㎖) + ADSC-derived exosomes (3.7x109/㎖), or psoriasis serum-derived exosomes (2.5 x 108/㎖) + ADSC-derived exosomes (3.7x109/㎖) for 24 h.
Nanoparticle tracking analysis
The size and number of extracellular vesicles (EVs) in conditioned medium were analyzed using NanoSight (NS300, Malvern, UK) with a 488 nm laser. The sample was loaded into the chamber using a 1 mL disposable syringe, and a 40-second video was recorded to capture all events. The video was then analyzed using NTA software based on Brownian motion. EVs were diluted with PBS until the appropriate number of EVs was detected, and measurements were taken at the same camera level and detection threshold. The analysis was repeated four times for each sample, providing data on the number and size distribution of exosomes.
Exosome uptake assay
HaCaT cells treated with psoriasis serum exosomes or stem cell exosomes were labeled with a green fluorescent dye, Vybrant DiO cell-labeling solution (Sigma‒Aldrich, USA). Cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 in 1% bovine serum albumin (BSA) for 10 minutes, and blocked with 3% BSA for one hour at room temperature. Cell-labeling solution was added to HaCaT cells treated with psoriasis serum exosomes or stem cell exosomes and incubated at 37°C for 2 hours. The cells were visualized under fluorescence microscopy using Leica microsystems DFi8 LASX software light microscopy (Leica, Wetzlar, Germany).
Quantitative PCR
Total RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. A First Strand cDNA synthesis kit (Roche Applied Science, Mannheim, Germany) was used to synthesize cDNA from 1 µg of total RNA. Quantitative reverse transcriptase-PCR (qRT‒PCR) was performed in triplicate using TaqMan master mix (Applied Biosystems, Foster City, CA, USA) and Real-Time PCR System (Applied Biosystems). The primer details for mRNA detection can be found in Supplementary Table 1. mRNA levels of IL-1β, IL-6, TNF-α, NOX2, NOX4, and Nrf2 were normalized to GAPDH. Fold changes were calculated using the ΔΔCt method, and relative quantification was performed using a Light Cycler 96 instrument (Roche Diagnostics, Mannheim, Germany).
Western blot assay
Cells were lysed in pro-prep lysis buffer (Intron, Seoul, Korea) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Protein concentrations were measured using a bicinchoninic acid solution with copper (II) sulfate (Sigma‒Aldrich, St. Louis, MO, USA). Equal amounts of protein (20 µg) were separated by 10% SDS‒PAGE, transferred to ECL nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK), and blocked for 1 hour with 5% skim milk in TBST. Membranes were incubated overnight at 4°C with the antibodies listed in Supplementary Table 2. Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, 1:1000; Abcam, Cambridge, UK) and chemiluminescent luminol (LUMINOGRAPH II; Atto, Tokyo, Japan). Immunocomplexes were visualized using an enhanced horseradish peroxidase/luminol chemiluminescence system (ECL Plus; Amersham International PLC, Little Chalfont, UK). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control for western blots.
Cell immunofluorescence
HaCaT cells were seeded on coverslips and allowed to adhere overnight. Cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 in 1% BSA for 10 min, blocked with 3% BSA for one hour at room temperature and incubated overnight at 4 ℃ with anti-LC3B (Abcam, Cambridge, UK). Cells were then incubated in goat anti-Rb HRP (Abcam, Cambridge, UK) and the secondary antibody conjugated with FITC (Abcam, Cambridge, UK) at 1:200 for 1 h. Stained HPDFs were captured and visualized using a microscope (Leica Micro systems, Germany). For nuclear counterstaining, Vectashield mounting medium was used along with DAPI (Vector Laboratories, Burlingame, CA).
Flow cytometry
Passage 4 and 7 ADSCs were detached with 0.05% trypsin/EDTA (Welgene, Gyeongsan, Gyeongsang, Korea; LS015-01) at 37°C for 3 min and washed using FACS buffer (1% BSA in PBS). The cells were bloked using FcR blocking reagent (Miltenyi Biotec, Auburn, CA, USA; 130-059-901) and stained with FITC-labeled antibodies against CD34 (BD Biosciences, San Diego, CA, USA; 555821) and CD29 (Dako, Glostrup, Copenhagen, Denmark; F7068) for 1 hour at RT. Fluorescence was analyzed using a BD FACSCalibur Flow Cytometer (BD bioscience, Heidelberg, Baden-Württemberg, Germany; 342975), and the data were analyzed by BD Cell Quest Pro software.
Statistical Analyses
Statistical analyses were conducted with GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA). Data were analyzed using the Student’s t-test and one-way analysis of variance with Tukey’s post-hoc test. P values < 0.05 were considered statistically significant.