Fig. 4

Anti-TNF-α therapy decreases NETosis. a Clinical and laboratory parameters, and (b) circulating levels of cell-free NETosis-derived products at baseline and after 6 months of infliximab (IFX) therapy. c-d Relative PAD4 and CitH3 protein expression at baseline and after IFX therapy. Representative blots are shown. Lanes were run on the same gel but non-contiguous. Cropping lines are used in the figure. e-h To assess the effect of IFX on NET formation, HD neutrophils (n = 3) were treated with TNF-α (8 ng/mL) in the presence or absence of IFX (100 mg/ml) for 2 h. e Representative scanning electron micrographs of NETs are shown in upper panels. Scale bar 10 μm. Fluorescence micrographs of NETs stained with Sytox dye or with neutrophil elastase (NE) antibody and DAPI are represented in middle and lower panels, respectively. Images were captured at 20x magnification. Scale bar 100 μm. f NET formation. Results were expressed as percentage of NETs. g Supernatant cell-free DNA levels. Data are represented as relative fluorescence units (RFU). h % NE-staining cells. a-h Bar graphs show the mean ± SEM. The data were analyzed using a paired-samples t test. *vs. baseline, #vs. TNF-α (P < 0.05). AU, arbitrary units; BASDAI indicates Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functionality Index; CitH3, citrullinated histone H3; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; HD, healthy donor; NETs, neutrophil extracellular traps; PAD4, peptidylarginine deiminase 4; r-axSpA, radiographic axial spondyloarthritis; TNF, tumor necrosis factor