Igg from human serum

For high content imaging experiments THP-1 cells were seeded into Microclear bottom 384-well plates (Greiner) at a density of 5000 cells / well and treated with 100 nM PMA for 24 h after which compounds were added at concentrations indicated in the corresponding section. On the third day PMA / compound medium was washed out and cells were cultured in compound-supplemented medium and stimulated with a combination of IL13 and IL4 at 25 ng / ml each for 48 h to induce M2 polarization. Afterwards, the FcRs-unspecific binding was blocked using 100 μg / ml IgG from human serum (Sigma-Aldrich), and M2 cells were stained with CD209-AlexaFluor647 (9E9A8). Subsequently, cells were fixed with FluoroFix buffer (Biolegend) and ultimately stained with Hoechst (Life Technologies). Images were obtained with the IN Cell Analyzer 2000 (GE Healthcare Life Sciences) and analyzed with Cell Profiler version 2.1.1 [37 (link)]. The median cell number analyzed in each well was 584. The signal was normalized to the corresponding isotype-stained DMSO controls and an intensity threshold was determined to bin CD209 positive and negative cells. The raw data obtained after image analysis is in S1 Table. Representative images shown were processed with FIJI [38 (link)] and the CD209-AlexaFluor647 staining was pseudo-colored yellow for better visualization.

Flow cytometry data was acquired using a CyAn ADP Analyzer (Beckman Coulter) and BD FACSAria II (BD) flow cytometers. FcRs-unspecific binding was blocked by incubating cells in 100 μg / ml IgG from human serum (Sigma-Aldrich). Cells were suspended in Cell Staining Buffer (BioLegend) and incubated with monoclonal antibodies. For the flow cytometric analysis of Cas9-FLAG expression, THP-1 cells were fixed and permeabilized with the BD Cytofix / Cytoperm (BD Biosciences) according to the manufacturer protocol and stained with the anti-FLAG antibody. Fluorescence was compared to the corresponding isotype-stained controls. All data were analyzed using the FlowJo software (Tree Star Inc.).

Phosphate-buffered saline (PBS) tablets (dissolved in deionized water: 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25 °C), Protein A from Staphylococcus aureus, and IgG from human serum were purchased from Sigma-Aldrich (Sigma-Aldrich Química S.L.U., Madrid, Spain).

Supernatants were collected from 5 × 10⁴ (Jaumouillé & Grinstein, 2011 (link)) macrophages incubated for 20 h on chambered slides coated with 0.01% poly‐L‐lysine (PLL) and 0–100 μg/ml IgG from human serum (Sigma Aldrich) or bilayers generated using 0–100 μg/ml IgG from human serum, centrifuged at 350 × g for 10 min and supernatants retained. The concentration of TNFα and IL‐10 was measured using enzyme‐linked immunosorbent assays (DuoSet, R & D Systems), according to the manufacturer's protocol.

The expression levels of CD14 in the THP-1 cell line, resting M0 and M1 macrophages were assessed using flow cytometry (FC). For FC, 2 × 10⁵ cells were detached, fixed, incubated with IgG from human serum (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) for 20 mins followed by incubation with anti-CD14 FITC mouse monoclonal antibody for 45 mins at room temperature in dark and washed twice with PBS. Flow cytometry analyzes were performed on samples using the Flow cytometer FC500 (Beckman Coulter, Brea, CA, USA).

For moDC surface marker analysis of the costimulatory and inhibitory molecules, moDCs (2 × 10⁵) were seeded in a 96-well plate and stimulated with 3 μg/mL Sa lysate or 100 ng/mL LPS with or without PSMα3 (10 μM) or PSMα3 alone for 6 h or 24 h. Cells were removed from the plate using Accutase (Sigma-Aldrich) and treated with IgG from human serum (1 μg of human IgG per 100,000 cells; Sigma-Aldrich) for 20 min at room temperature to avoid unspecific binding via Fc receptors. Cells were stained with ZombieAqua (BioLegend) to exclude dead cells and fluorochrome conjugated extracellular antibodies: HLA-DR BV650 (L243, BioLegend), CD11b BV510 (ICRF44, BioLegend), CD11c APC (MJ4-27G121, Miltenyi), CD11c PE-Cy7 (Bu15, BioLegend), CD40 FITC (5C3, eBioscience), CD80 PE-Cy7 (2D10, BioLegend), CD83 PE-Dazzle 594 (HB15e, BioLegend), CD86 BV605 (IT2.2, BioLegend), PD-L1 PE (29E.2A3, BioLegend), PD-L2 PE (MIH18), ILT3 PE (ZM4.1, BioLegend) for 20 min at 4°C. FACS buffer [PBS containing 1% FBS, 2 mM EDTA (Merck) and 0.09% NaN₃ (Sigma-Aldrich)] was used for all incubations and washing steps. At least 50,000 cells were acquired using a LSR Fortessa flow cytometer (BD Biosciences) with the DIVA software (BD Biosciences) and were further analyzed using FlowJo 10.4.2 software (Tree Star).