Fig. 2: IL-33 activates RA neutrophils to increase NET generation.

a NET–DNA levels produced from RA neutrophils incubated with various concentrations of IL-33 for 4 h. b Dynamic observation of NET formation in RA neutrophils induced with 100 ng/mL IL-33 using a live cell workstation. c Representative immunofluorescence staining and scanning electron microscopy images of NET formation induced by 100 ng/mL IL-33 in RA neutrophils. d Cellular ROS levels in neutrophils stimulated with 100 ng/mL IL-33, 40 nM PMA, or PBS for 2 h. e NET–DNA levels produced by RA neutrophils pretreated with 10 μM DPI, 10 μM Cl-amidine or 20 μM sivelestat for 1 h prior to stimulation with 100 ng/mL IL-33 or 40 nM PMA for 4 h. f Coimmunoprecipitation assay showing interactions among ST2, MyD88, and TRAF6 in RA neutrophils treated with 100 ng/mL IL-33 for 1 h. g Representative western blots showing ERK1/2, p38, IκBα and p65 phosphorylation levels in RA neutrophils incubated with 100 ng/mL IL-33 for 4 h. h NET–DNA levels induced from RA or HD neutrophils pretreated with 0.2 mg/mL ST2 polyclonal neutralizing antibody for 1 h and then stimulated with 100 ng/mL IL-33 or 40 nM PMA for 4 h. i ST2 mRNA levels in peripheral blood neutrophils from RA patients (n = 18) and HDs (n = 18) with or without incubation with 100 ng/mL IL-33 for 12 h. j Flow cytometric analysis of the proportion of CD66b+ST2+ cells among peripheral blood neutrophils from RA patients (n = 18) and HDs (n = 18) with or without incubation with 100 ng/mL IL-33 for 12 h. k, l Fold change in the ST2 mRNA level and the proportion of CD66b+ST2+ cells among RA (n = 18) or HD (n = 18) neutrophils incubated with 100 ng/mL IL-33 for 12 h. All the data are presented as the means ± SDs/SEMs. Asterisks represent significant differences compared with the reference group, which is without asterisks. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant.