Il 12p70

Luminex, model L200 (Millipore, Massachusetts, USA) was used to evaluate serum levels of, IL-8, IL-12p40, IL-12p70, VEGF-A, MMP-1, and-8 (R&D Systems, Minneapolis-USA). The results were presented in median fluorescence intensity (MFI) units.
Enzyme-linked immunosorbent assay (ELISA) was used to evaluate serum levels of citrullinated histone H3 (Cayman, Michigan-USA) and alpha-1-antitrypsin (Abcam, Cambridge-USA).

Standard ELISA methods were used to measure concentrations of Gal-9 (R&D Systems), cytokines and chemokines such as IL-12p40, IL-12p70, MIP-1β (macrophage inflammatory protein [MIP]), monocyte chemoattractant protein 2 (MCP-2), tumour necrosis factor-alpha (TNF-α), IL-6, transforming growth factor-beta1 (TGF-β1), IL-18, IL-23, IL-27, and IL-1β (R&D Systems) and IFN-α (Verikine™ Human IFNα ELISA Kit, PBL Assay Science, Piscataway, NJ, USA).

Differentiation of murine T cells was performed as previously described (40 (link)). Splenic T cells were isolated by magnetic activated cell sorting using the “pan T cell isolation kit II” according to the manufacturer’s instructions (Miltenyi Biotech). Cells were fluorescently stained with an antibody cocktail containing αCD4-FITC (RM4-5, eBioscience), αCD44-PE (IM7, Biolegend), αCD62L-APC (MEL-14, eBioscience), and αCD25-PE-Cy5 (PC61.5, eBioscience) and were subsequently isolated by fluorescence activated cell sorting on MoFlo (Beckman-Coulter, Brea, CA, USA) in the FACS-core unit of the University Hospital Erlangen. Sorted naive T cells (CD4⁺CD62L⁺CD44^lowCD25⁻) were stimulated by plate-bound anti-CD3 (2 µg/ml, 145-2C11, BD Pharmingen, San Diego, CA, USA) and soluble anti-CD28 (2 µg/ml, 37.51, BD Pharmingen) and cultured for 4 days in the presence of IL-6 (40 ng/ml, R&D Systems, Minneapolis, MN, USA) and TGFβ1 (2 ng/ml, Biolegend) for Th17, IL-12p70 (20 ng/ml,R&D Systems) and anti-IL-4 (10 µg/ml, Biolegend) for Th1 or TGFβ1 (10 ng/ml, Biolegend) alone for Treg differentiation. T cell frequencies were analyzed by flow cytometry (FACSCantoII, BD Biosciences) using the antibodies listed in the section “Murine FACS analysis.”

Soluble biomarkers were measured using multiplex magnetic bead immunoassay based on Luminex® xMAP multianalyte profiling platform and analyzed on MAGPIX® System (Merck Millipore) at the Institute of Medical Biochemistry and Laboratory Diagnostics of The General University Hospital and of the First Faculty of Medicine of Charles University in Prague. The following premixed magnetic bead kits were used for plasma sample analysis: LXSAHM-07: endoglin (BR22), IL-1β (BR28), IL-22 (BR35), IL-6 (BR13), procalcitonin (BR39), VCAM-1 (BR57), and VEGF-A (BR26) and LXSAHM-08: angiopoietin-2 (BR26), endothelin-1 (BR76), ICAM-1 (BR61), IL-1α (BR38), IL-17A (BR42), IL-18 (BR78), IL12 p70 (BR563), TNF-α (BR12) (both from Biotechne R&D Systems), and Milliplex MAP human soluble receptor magnetic bead panel (HSCRMAG-32K-02: VEGFR1 and VEGFR2) and cardiovascular disease (CVD) magnetic bead panel (HCVD1MAG-67K-01: endocan1/ESM1) (Merck).

Concentrations of IL15/IL15R, IL12p70 and TNFα (R&D Systems) as well as IFNγ (Pharmingen) in serum and cell supernatant were determined by ELISA according to the manufacturers’ protocol.

Cell-free supernatants of infected DCs or co-cultures were collected at 48 h to measure secretion of IFN-α (Thermo Fisher Scientific, Waltham, MA, USA), IFN-β, IFN-γ, TNF-α, IL-10, and IL-12p70 (R&D Systems, Minneapolis, MN, USA) using ELISA kits according to the manufacturers’ instructions.