CONICET | Buscador de Institutos y Recursos Humanos

Neutrophils mediate bacterial killing by either phagocytosis or the release of extracellular traps (NETs). NETs are structures made of chromatin associated with specific proteins from the granules, that contribute to the extracellular elimination of pathogens. The airway epithelium is constantly interacting with the environment, and it was found to express several pattern recognition receptors such as the DNA receptor TLR9. When these receptors are stimulated they lead to the production of cytokines and chemokines that promote inflammation and recruit phagocytes to clear the infection. In this inflammatory context neutrophils can transmigrate the pulmonary epithelium and release NET, as has been reported in pulmonary alveoli of mice infected with S. pneumoniae. The aim of this study was to address if NETs exert immunomudulatory effects on epithelial cells. NETs were obtained from 2 x 106/ml neutrophils isolated from peripheral blood from healthy donors cultured with MSU crystals (300 μg/ml) for 4 h at 37° C. As controls, NETs were treated with micrococcal nuclease (MNase, 10 U/ml) and cytoplasms were obtained from 2 x 106/ml neutrophils by heating at 45o C and cooling. A549 epithelial cells at 80-90% confluence were incubated with NETs, MNase-treated NETs, neutrophil cytoplasms or MNase for 24 h at 37o C with or without LPS (250 ng/ml). Culture supernatants were collected and IL-6 and MCP-1 were determined by ELISA. NETs or MNase-treated NETs significantly increased the secretion of IL-6 and MCP-1 by epithelial cells either in the absence or presence of LPS (IL-6, p < 0,05 n=4; MCP-1, p < 0,05 n=4;) whereas neutrophil cytoplasms had no stimulatory effects. None of the treatments affected the viability of A549 cells as assessed by 7-AAD uptake and flow cytometry. Since the effect of NETs on epithelial cells was not modified by MNase treatment, it cannot be attributed to DNA but might be mediated by histones, a hypothesis that we are currently testing.