Legendplex panels

Cytokines present in the induced sputum supernatant were analyzed, using LEGENDplex panels (BioLegend, San Diego, CA, USA), to determine levels of the following: eotaxin; immunoglobulins IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM; interferon alpha and gamma (IFNα and IFNɣ); interleukins IL-1Β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-23, and IL-33; monocyte chemoattractant protein 1 (MCP-1); matrix metalloproteinase 2 and 9 (MMP2 and MMP9); and tumor necrosis factor alpha (TNFα).

The cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.25% SDS, 10% Glycerol, 1% NP-40 and one tablet of complete protease inhibitors) for 15 min on ice. Lysates were analyzed on SDS-polyacrylamide gels (SDS-PAGE; 12-14%), along with protein molecular weight markers (Nippon MWP03, Fermentas, Thermo Scientific, SM0431) and blotted onto nitrocellulose membrane (GE Healthcare). After blocking with 5% milk in TBS-Tween 20 buffer, membranes were incubated with primary and HRP-conjugated secondary antibodies (Biotech, anti-rabbit HRP 1030-05 or anti-mouse HRP 4050-05) signals were visualized by enhanced chemiluminescence (ECL, GE Healthcare) using films or a ChemiDocTMXRS+ System with Image LabTM software. Primary antibodies used: anti-AUF1 (milipore, 07-260) and anti-GAPDH (Ambion, 6C5). Cell culture Supernatants or serum were analyzed via specific ELISA kits of TNFa, IL-10, IL-6, VEGF, IL12 (Peprotech 900-K54, 900-K53, 900-K50, 900-K99), IL-1b (Thermo-Fisher, 88-7013-88), Anti-Ovalbumin IgE (Cayman 500840) or cytometric bead-based assay panels following manufacturer’s instructions Legendplex Panels (Biolegend, Cat. No.741044 and 740846). Protein levels were normalized to cell number using crystal violet staining.

Secreted levels of human or mouse IL-17A in plasma or serum were determined according to the manufacturer’s instructions (LEGEND MAX Human IL-17A ELISA Kit, LEGEND MAX Mouse IL-17A ELISA Kit, BioLegend). For human samples, plasma samples from patients with psoriasis were used as internal reference controls. For multiplex quantification of cytokines, the bead-based LEGENDplex panels (Human Th17 7-plex Panel; Human Th 12-plex Panel, Mouse Th17 7-plex Panel; IL-1β, IL-23 and IL-12p70 from the Inflammation Panel 1; granzyme A and granzyme B from the CD8/NK Panel, predefined and custom-designed mix-and-match system from BioLegend) were used according to the manufacturer’s instructions. Flow cytometry reading was performed on the FACSAria III (BD). Mean fluorescence intensity values were recorded using LEGENDplex analysis software (version 2021.07.01), and cytokine concentrations (pg ml⁻¹) were interpolated from a five-parameter logistic non-linear curve model using a separate standard curve for each cytokine. For prognostic stratification of IL-17A plasma levels, an optimal cut point was determined in each dataset separately using X-tile^{28 (link)}.