Standard protocol approvals, registrations, and patient consents
This is a retrospective case series study of CRPS patients seen at the Drexel University College of Medicine pain clinic that met the Budapest consensus criteria for CRPS and received PE as treatment for their illness between September 2012 and June 2014. All participants agreed to provide blood samples for this study after giving informed consent as approved by the institutional review board.
Plasma exchange and patient evaluation
The patients were asked to provide blood samples before and after PE. Patients with autoimmune or immunodeficiency conditions were excluded. Patient records were reviewed from which data regarding demographics, CRPS signs and symptoms, duration of illness and response to PE were obtained. PE was performed in patients with refractory CRPS over 2 weeks at the Hahnemann University Hospital, Philadelphia. All patients had full cardiac and neuropsychological clearance before PE. Patients were asked to rate their overall pain before, during, and after the apheresis using a 11-point numerical rating scale (NRS) from 0 (no pain) to 10 (the worst pain imaginable).
Characterization of exosomes by nanoparticle tracking analysis, electron microscopy and western blot analysis
One milliliter of serum from CRPS patients was diluted in equal volume of PBS and centrifuged at 2000×g for 30 min at 4 °C. The supernatant was diluted to final volume of 24 ml in 1× PBS and centrifuged at 12,000×g for 45 min at 4 °C. The supernatant was filtered through a 0.22 µ filter and centrifuged at 110,000×g for 70 min at 4 °C. The exosome pellet obtained was washed in 25 ml 1× PBS without ions and centrifuged at 110,000×g for 70 min at 4 °C. The exosome pellet was resuspended in 100 µl of PBS for use in nanoparticle tracking analysis, electron microscopy and protein estimation.
Nanoparticle tracking analysis
Exosomes in PBS were analyzed for size and concentration using the NanoSight NS300 according to the manufacturer’s protocol (Malvern Instruments, MA, USA). Samples were diluted to ~ 107–109 particles/ml and continuously injected with a syringe pump and three videos (30 s each) were captured for particle analysis. Nanoparticle tracking analysis was performed using NTA 3.2 software.
Ten microliters of PBS resuspended exosomes were coated on Ni-formvar grids and incubated for 20 min at RT. The grids were washed on 50 µl drops of 0.1 M Sorensen’s phosphate buffer (pH 7.2) for 5 s each for a total of five times. The grids were blot dried perpendicularly on whatman #1 filter paper. Negative staining and embedding were performed by incubating the grids on 0.5% uranyl acetate (in a 0.2% methyl cellulose solution) for 10 min at 4 °C. The excess solution was blotted on a Whatman paper, air dried and imaged in a JEOL Transmission Electron Microscope (JEM 1230). Alternatively, the exosomes were immunolabelled for the exosome marker CD81 and crosslinked with 1% glutaraldehyde, and probed with 6 nm gold secondary antibody, followed by negative staining and embedding.
Protein concentration was estimated using a DC Protein assay (Bio-Rad Laboratories, CA, USA); 5 µg of exosomes isolated from CRPS serum were resolved on a reducing 12% SDS-PAGE, transferred to PVDF membrane and blocked in Odyssey Blocking buffer (927-50100, LI-COR Biosciences) for 2 h. The membrane was incubated in rabbit anti-CD63 antibody (ab68418, Abcam) overnight at 4 °C, washed thrice in TBST, 10 min each, and incubated in goat anti-rabbit 680RD IgG (925-68071, LI-COR Biosciences) for 45 min at RT, washed thrice in TBST for 10 min each, and imaged on an Odyssey Fc imaging system.
Exosome miRNA profiling
RNA was isolated from exosomes using a miRvana miRNA isolation kit (Life technologies) following the manufacturer’s protocol. Taqman low-density array microfluidic cards version A and B (Applied Biosystems, Foster City, CA) were used to profile miRNAs in 100 ng of total RNA as previously described . miRNA species with CT values 35 and higher were treated as undetected. Fold change was calculated from raw CT values using the 2−ΔΔCT method . The mean CT values of the 10 miRNAs with the lowest standard deviations across all samples were used as the endogenous control in the calculation of ΔCT. Statistical significance of differences in ΔCT values was calculated by a two-tailed paired t-test for comparison of pre- and post-PE samples and by a two-tailed independent t-test for comparison of other experimental groups. A p-value threshold of 0.05 and a fold-change of 2 were used to select significant differentially expressed miRNAs between experimental groups.
HEK293 cells obtained from the American Type Culture Collection (ATCC) was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. Human monocytic THP-1 cells (TIB-202, ATCC) cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS).
Luciferase reporter assay
The 3′UTR luciferase reporter constructs for IL-6 (NM_000600.2) was purchased from GeneCopoeia. The miRNA mimics for human miR-338-5p (MC12825), anti-miR-338 (MH12825) and scrambled negative control (4464058) was purchased from Life technologies. HEK293 cells were co-transfected with mir-338-5p, anti-miR-338 or miRNA scrambled control and luciferase reporter plasmid containing the 3′UTR of human IL-6 using Lipofectamine LTX (Life Technologies, Carlsbad, CA) for 48 h. The Luc-Pair miR Luciferase assay kit (GeneCopoeia) was used to measure firefly and Renilla luciferase activity according to the manufacturer’s instruction. Firefly luciferase measurements normalized to Renilla luciferase was used as a transfection control. The data expressed as percentage of control is the average of three independent experiments.
miR-338-5p overexpression in THP-1 cells
Transfections were performed following the manufacturer’s protocol for RNAiMax transfection reagent using either miR-338-5p or negative control with the following modifications. For each well of a 6-well plate, 7.5 µl RNAiMax reagent was diluted in 150 µl of serum-free media, and 30 pmol of miR-338-5p or control mimic was diluted in 150 µl of serum-free media individually. The dilutions were combined and incubated at room temperature for 15 min. This transfection complex (300 µl) was added to 0.5 × 106 cells/well in 1.7 ml serum containing media in 6-well plates and incubated for 6 h at 37 °C, after which the media was changed. After 24 h, cells were treated with 1 µg/ml lipopolysaccharide (LPS) in complete culture media for 6 h. Exosome deplete media was used in all experiments. Cells were collected by centrifugation at 135×g for 5 min at 4 °C and the conditioned media was stored at 4 °C. The cell pellet was washed with 1× PBS and resuspended in either RNA lysis buffer (mirVana kit; Life Technologies) containing 0.5 U/µl RNAsin Plus (Promega; Madison, WI) for RNA isolation or 1× radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Thermo Scientific; Waltham, MA) for protein analysis.
cDNA synthesis and qPCR for mRNAs
mRNA was isolated using the miRVana kit (Life technologies). The Maxima cDNA synthesis kit (Thermo Scientific) was used to generate cDNA and 2 µl cDNA was used for Taqman based quantitative real time mRNA analysis containing 10 µl Taqman Fast Universal polymerase chain reaction (PCR) master mix (2×) no AmpErase UNG (Life Technologies), 1 µl Taqman primer–probe (20×), in up to 20 µl nuclease-free water. GAPDH was used as the normalizer and one-way ANOVA was used to perform statistical analysis. Assay IDs were as follows: Hs00985639_m1 [IL-6], 4325792 (GAPDH) (Applied Biosystems, Carlsbad, CA).
Enzyme-linked immunosorbent assay (ELISA) for IL-6 in cell culture media
Supernatants collected after miR-338-5p or control miRNA transfections in THP-1 cells were used to perform ELISA for secreted IL-6 using the human IL-6 quantikine ELISA kit (D6050) according to the manufacturer’s protocol (R&D Systems; Minneapolis, MN).
Cytokine measurement in patient plasma
For plasma isolation, blood was collected into EDTA-coated (purple top) vacutainers. The plasma was separated by centrifugation (3000×g for 15 min at 4 °C), split into 250 µl aliquots and stored at − 70 °C until assayed. The MILLIPLEX MAP Human High Sensitivity T Cell Panel, HSTCMAG-28SK (Millipore, Billerica, MA) was used to determine plasma levels of 14 cytokines. The minimum detectable concentration for IL-6 and TNF-α was 0.11 and 0.16 pg/ml respectively. All assays were performed in duplicate according to the manufacturers’ instructions. Assay results were determined on a Luminex-200 (Luminex, Austin, TX).
Data are presented as mean ± the standard error of the mean from three or more independent experiments. Student t-test was used for determining the statistical significance. Treatment effects were analyzed with a one-way analysis of variance (ANOVA). Pairwise comparisons between means were tested using the post hoc Dunnet method. Error probabilities of p < 0.05 were considered statistically significant. Paired t-test with Bonferroni correction for multiple assays was used for analysis of the cytokine panel. Error probabilities of p < 0.00357 were considered statistically significant.