Biomarker Analysis

Blood samples were collected using tubes containing ethylene diamine tetraacetic acid (EDTA) from all participants between 8 am and 3 pm and processed within 4 h of collection. All samples were centrifuged at 3000 rpm for 10 min (4°C) and plasma was aliquoted and stored at −80°C until analyzed. For each participant, 0.5 ml of frozen plasma were used to isolate EVs. Thrombin was added to thawed samples, incubated at room temperature for 5–10 min and then centrifuged for 5 min at 10,000 rpm. The supernatant was removed from each sample and ExoQuick (System Biosciences) solution was added according to manufacturer’s instructions. After a 30-min incubation at 4°C, samples were centrifuged at 1,500 g for 30 min. The supernatant was aspirated resulting in an EV pellet at the bottom of the tube. The pellet was resuspended in 500 µL phosphate-buffered saline. We used a human tumor susceptibility gene 101 protein (TSG101, EV marker) ELISA kit to confirm the presence of EVs in the samples (CSBEL025125HU, CUSABIO, Houston, TX) (Supplementary Table S1). For protein analysis, mammalian protein extraction reagent (M-PER) was added to each tube to lyse EVs (Thermo Scientific, Inc., Rockford, IL).

Plasma and EV Levels of IL-6, IL-10 and TNFα were measured using an ultrasensitive assay (Neurology 3-Plex A, item 101995, Quanterix Corporation, Lexington, MA) and single-molecule technology (SIMOA™) on a HD-1 analyzer (Quanterix Corporation) and reported in pg/mL. Samples were randomized over plates and tested in duplicate with laboratory scientists blinded to participant groups. All samples underwent quality control analysis and samples with reported coefficients of variation over 20% were not used for analysis. Samples with high coefficients of variation and concentrations below lower limits of quantification were replaced by values equal to half of the limit of concentration respective to the assay.