Fig 2: Flow cytometry with gating strategy depicted confirms 98.1% purity of CD66b/CD16 double-positive neutrophils.

Fig 3: CF sputum supernatant treatment does not affect PMN viability. PMN purity and viability following isolation from blood and subsequent 3.5 h-CF sputum incubation was measured by flow cytometry using the Zombie Aqua Cell Viability Kit™. (A) Representative images of the gating strategy used to determine the percent of viable PMNs (CD66b+/Zombie Aqua-) for each condition tested are shown. (B) Flow cytometric analysis showed no significant difference in the percent of PMNs and CD66 surface expression among the conditions tested (n = 15). (C) CF sputum treatment does not impair PMN viability under the conditions used in this study. Treatment with 100 nM PMA for 30 min, however, results in a significant decrease in PMN viability compared to both the sputum supernatant-treated and untreated PMNs (n = 15). One-way ANOVA, Tukey’s multiple comparison test. **, p < 0.01; ns, not significant. PMA, phorbol 12-myristate 13-acetate; CF SS; Cystic fibrosis sputum supernatant.

Fig 4: Immune response profile of FN- and DCN-coated planar scaffolds: (a) Schematic overview of the analysis steps and used immune cell assays. Polymorph nuclear cells (PMNs) and peripheral blood mononuclear cells (PBMCs) were isolated from human blood. Monocytes were acquired from PBMCs by magnetic separation via CD14 beads. Monocytes were differentiated into M0 macrophages (MØ) by stimulation with 50 ng/mL of macrophage colony-stimulating factor (M-CSF) for 7 days. (b) Surface expression of activation markers CD11b and CD66b by PMNs after 4 h: Displayed are the mean fluorescence intensities (MFI) normalized to unstimulated PMNs after isolation as mean ± SEM (standard error of the mean) for unstimulated (unstim) and lipopolysaccharide (LPS)-stimulated cells, as well as PMNs cultured on the uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds determined with flow cytometry. Kruskal–Wallis test, n = 6. (c) Surface expression of activation markers CD80 and human leukocyte antigen DR isotype (HLA-DR), and tumor necrosis factor alpha (TNFa) release by monocytes. Shown are the MFI normalized to unstimulated monocytes as mean ± SEM for LPS-stimulated cells as well as monocytes cultured on uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds. Kruskal–Wallis test, n = 6–8. The TNF release is depicted in ng/105 cells as mean ± SEM for unstimulated (unstim) and LPS-stimulated cells as well as monocytes cultured on the uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds. Kruskal–Wallis test, n = 5. (d) Surface expression of activation markers CD80 and HLA-DR, and TNFa release by macrophage: Displayed is the MFI normalized to unstimulated M0 macrophages as mean ± SEM for macrophages differentiated to M1 and as well as cells cultured on uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds. Kruskal–Wallis test, n = 6–8. The TNFa release is shown in ng/105 cells as mean ± SEM for unstimulated M0 macrophages; macrophages differentiated to M1; and as well as cells cultured on the uncoated (w/o), DCN-coated (DCN), and FN-coated (FN) scaffolds. Kruskal–Wallis test, n = 6–9. (e) Representative SEM images of monocytes (left) and macrophages (right) on uncoated (w/o) and with biofunctionalized scaffolds (DCN and FN). Scale bars represent 50 µm. (f) Expression of activation markers CD69, CD25, and HLA-DR on CD3+ T cells in whole PBMC co-cultures: Shown are representative histograms (left) and the surface expression levels as MFI normalized to unstimulated T cells as mean ± SEM (right) for aCD3/aCD28-stimulated T cells (stim) as well as T cells cultured on uncoated (w/o), DCN-coated, and FN-coated scaffold. Kruskal–Wallis test, n = 6.

Fig 5: Glucocorticoid treatment amplified the percentages of MDSC and the two subsets in PBMC of relapsing MS patients. A, Staining profiles of MDSC (CD11b+CD33+HLA-DR-), M-MDSC (CD11b+CD33+HLA-DR-CD14+CD66b-) and G-MDSC (CD11b+CD33+HLA-DR-CD14-CD66b+) from a representative MS patient before and after methylprednisolone impulse treatment. B, Percentages of MDSC (P < 0.0001), G-MDSC (P = 0.0001) and M-MDSC (P = 0.0024) in PBMC of MS patients (n = 12) before and after treatment. C, Percentages of G-MDSC (P = 0.0035) and M-MDSC (P = 0.0096) in MDSC of PBMC from MS patients (n = 12) before and after treatment. D, Naïve human CD4+ T cells were stimulated by anti-CD3/CD28 in the absence or presence of MDSC from MS patients for 3 d (G-MDSC, P = 0.0005, n = 3; M-MDSC, P < 0.0001, n = 3; G-MDSC vs M-MDSC, P = 0.3701, n = 3), and T-cell proliferation was determined by measuring BrdU incorporation (Brdu was added 6 h prior to cell harvest)