Immunomodulatory effects of neutrophil extracelular traps (NETS)

SABBIONE, FLORENCIA; KEITELMAN, IRENE; IULA, LEONARDO; PODAZA, ENRIQUE A.; TOWSTYKA, NADIA YASMÍN; GIORDANO, MIRTA; CAROLINA, JANCIC; ANALÍA, TREVANI

Mar del Plara

Congreso; 62 Reunion Anual de la Sociedad Argentina de Inmunologia; 2014

Sociedad Argentina de Inmunologia

Airway epithelial cells provide a mechanical barrier to prevent lung infection and produce chemokines and cytokines that recruit and activate phagocytic cells. In response to alveolar inflammatory stimuli, neutrophils transmigrate the epithelium to eliminate microorganisms by either phagocytosis or NETs. They are chromatin fibers decorated with antimicrobial proteins that can trap and kill microorganisms. The aim of this study was to address if NETs also exert immunomodulatory actions. NETs were obtained from culture supernatants of neutrophils isolated from healthy donors by stimulation with monosodium urate crystals (300 ìg/ml) for 4 h at 37° C, gentle sonication and centrifugation. As controls, neutrophils were also stimulated in the presence of Diphenyleneiodonium (DPI) or elastase inhibitor (EI) to avoid NETs release. The presence of NETs in supernatants was determined by fluorometry. NETs preparations or their controls were added to A549 and BEAS-2B epithelial cells cultures for 24 h at 37°C and then supernatants were collected and IL-6 and IL-8 were determined by ELISA. Cell viability was assessed by Annexin V-FITC/PI staining by FACs. NETs increased the secretion of IL-8 and IL-6 by both types of epithelial cells (n=3, p<0.0001) in contrast to supernatants of stimulated neutrophils treated with either DPI or EI, which induced the production of much more reduced levels of cytokines, similar to those induced by unstimulated neutrophil supernatants. None of these treatments affected cell viability. Cytokines levels produced by A549 cells in response to NETs were similar to those produced in response to LPS and PAM3CSK (n=5). The cytokine stimulatory effects mediated by NETs were also observed in macrophages differentiated from human monocytes (n=4 p<0,001), but not in monocyte derived-dendritic cells (n=5). Altogether our results suggest that beyond their microbicide properties, NETs also constitute potent and selective proinflamatory stimuli.