Sample preparation

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood using Ficoll gradient centrifugation (Histopaque Sigma) as previously described [18, 64]. Freshly isolated PBMCs were stimulated with 10 μg/ml of whole glucan particles (WGP Dispersable or 1,3/1,6-β-glucan) (InvivoGen, tlr-wgp) which is a specific Dectin-1 agonist that lacks TLR stimulating activity—for 18 hours (for Monocyte experiments), and 12 hours (for dendritic cell experiments) in RPMI medium supplemented with 10% fetal bovine serum and Penicillin/Streptomycin/L-Glutamine at 37° C. WGP (Soluble, or 1,3/1,6-β-glucan) (Invivogen tlr-wgps) was used as a negative control. WGP soluble particles can bind to the Dectin-1 receptor without activating it. Baseline samples were completely unstimulated. Brefeldin-A (GolgiPlug, BD Biosciences) was added for the last 6 hours of stimulation. Cells were then surface stained with anti-CD14-PE-CF594 (3G8, BD-Biosciences), anti-CD16-PE-Cy7 (3G8, BD-Biosciences), and anti-CD11b-APC-Cy7 (ICRF44, BD-Biosciences) to identify monocytes and separate them into activate, inflammatory, classical and non-classical subsets. Dendritic cells were identified by staining for lineage markers (CD3, CD14, CD16, CD19, PE-TXR, BD Biosciences) in a general dump gate. Lineage negative cells that were also anti-HLA DR+-APC-Cy7) (LN3, eBioscience) (Lin-/HLA DR+) were then stained for anti-CD11c-PE-Cy7 (3.9, eBioscience), and anti-CD123-APC (7G3, BD Biosciences) to separate dendritic cells into myeloid dendritic cells (CD11c+, CD123-), and plasmacytoid cells (CD11c-, CD123+). Cells were fixed in Cytofix buffer (BD-Biosciences) and stored at -80° C in freezing medium until analyzed using flow cytometry. On the day of analysis cells were thawed, washed, and permeabilized with Cytofix/Cytoperm (BD-Biosciences) and Perm/Wash buffer (BD-Biosciences) for intracellular cytokine staining with anti-interleukin-10 (IL-10) Pacific Blue (JES3-9d7, eBioscience), anti-interleukin-12 p70 (IL-12) PE (20C2, BD Biosciences), anti-interleukin-6 (IL-6) fluorescein isothiocyanate (MQ2-13A5, eBioscience), and anti-tumor necrosis factor-α (TNF-α) Alexafluor 700 (MAB11, BD Biosciences) for monocyte staining. Dendritic cells intracellular staining used the following antibodies: anti-IFN-α-FITC (pbl interferon source, 21112-3), anti-interleukin-12 p70 (IL-12) PE (20C2, BD -Biosciences), anti-IL-6-Alexaflour 700 (MQ2-13A5, eBioscience), anti-tumor necrosis factor-α (TNF-α)-Pacific blue (Mab11, eBioscience). Samples were run using a Fortessa LSRII flow cytometer (Becton Dickinson) and analyzed using FlowJo software (FlowJo, LLC). A general gating strategy is shown in Supplementary Figure 1. Cells were also analyzed for Dectin-1 surface expression (anti-Dectin-1, 15E2, eBioscience) by flow cytometry.