Brefeldin a bfa

To investigate the changes of Treg and Th17 cells in MMD patients, flow cytometry was used to measure the percentage of Th17 and Treg cells. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from the fresh blood by Ficoll gradient centrifugation. For Th17 cells, the isolated PBMCs were pre-treated with phorbol 12-myristate-13-acetate (PMA, 25 ng/ml), ionomycin (50 µg/ml) and brefeldin-A(BFA, 10 µg/ml, Enzo Life Sciences, Farmingdale, USA) at 37 °C for 4 h, then were collected and surface-stained with antibodies (eBiosicence, Frankfurk, Germany) at room temperature (RT) for 15 min, followed by fixed and permeabilized with the transcription factor staining buffer set kit according to the manufacturer’s protocol (eBioscience), then intracellular-stained with IL-17 antibody (eBioscience) for 30 min. Treg and its subtypes were incubated with surface antibodies at RT for 15 min. After fixed and permeabilized, cells were stained with Foxp3 antibody (eBiosicence) at RT for 30 min. Isotype controls were used in parallel. CD4⁺CD25⁺Foxp3⁺ cells were considered as Treg cells while CD3⁺CD8⁻IL-17⁺ cells were defined as Th17 cells. Cells were measured using flow cytometry (BD bioscience, San Diego, CA, USA). Data were analyzed using Flowjo software (Tree Star, Ashland, OR).

TLR activation of monocytes was analysed concerning induction IL-1beta, IL-6 and TNF-alpha. Thawed PBMC were incubated in RPMI1640 with and without Pam3CSK4, LPS-B5 and CpG-ODN-2216, respectively. After 4 h, brefeldin A (BFA, 0.5 μg/ml; Enzo Life Sciences GmbH, Lörrach, Germany) was added for additional 16 h. Then, dead and viable cells were discriminated by Zombie Aqua^TM staining (BioLegend, London, UK). After adding TruStain FcX® (BioLegend), cells were stained with anti-CD3 (APC-Cy7-labelled), anti-CD16 (FITC-labelled) and anti-CD14 (PerCP-labelled) (all BioLegend). Then, cells were fixed and permeabilized (Cytofix/Cytoperm Kit; BD Pharmingen) to enable intracellular staining with PE-labelled anti-IL-1beta (Life Technologies GmbH, Frankfurt, Germany), BV421-labelled anti-IL-6 and PE-Cy7-labelled anti-TNF-alpha (both BioLegend). Finally, samples were analysed on a FACSCanto II (BD Biosciences, Heidelberg, Germany) with the FlowJo V10 software (TreeStar Inc., Ashland, OR, USA). Percent IL-1beta-, IL-6- and TNF-alpha-positive cells measured in the classical (CD14⁺⁺CD16^–), intermediate (CD14⁺⁺CD16⁺) and non-classical (CD14⁺CD16⁺⁺) monocyte subsets (Supplementary Figure 1). Fluorescence minus one (FMO) controls and isotype controls were performed for all antibody panels to define positive signals and confirm proper compensation.