Fig. 3.

Evaluation of the agonistic capacity of NET to stimulate cytokine production by human epithelial cells. a–c Monolayers of A549 epithelial cells were stimulated with SN MSU-induced NET, the TLR4 agonist LPS from E. coli O111:B4 (250 ng/mL); the TLR2 agonists PAM2CSK4 (100 ng/mL) and PAM3CSK4 (1 µg/mL), or the TLR5 agonist, flagellin (1 µg/mL) for 18 h at 37°C. Cytokine concentrations were determined in culture supernatants by ELISA. d, e Polarized 16HBE14o- cell monolayers were apically stimulated for 18 h with SN unPMN, SN MSU-, or CSE-induced NET or flagellin (1 µg/mL), and the CXCL8/IL-8 concentrations in the culture supernatants were determined by ELISA. f Monolayers of A549 epithelial cells were stimulated for 18 h with SN MSU-induced NET in the presence or absence of LPS (250 ng/mL), and the IL-6 concentration in the culture supernatants was determined by ELISA. Data are depicted as the mean ± SEM of 5 independent experiments performed in triplicate. * p < 0.05 (two-way ANOVA with repeated measures, followed by Bonferroni's multiple-comparisons test).