Low concentrations of neutrophil extracellular traps induce proliferation in human keratinocytes via NF-kB activation

Granulocytes play a pivotal role in innate immune response, as pathogen invasion activates neutrophils, a subclass of granulocytes, inducing the production of neutrophil extracellular traps (NETs). In this study, it has been evaluated how NETs could affect human keratinocytes (HaCaT cells) behaviour.HaCaT cells were treated with increasing NETs concentrations (0.01-200ng/ml) and the effect on cell proliferation was evaluated by MTT assay. Inhibition studies were performed by pre-treating cells with dexamethasone, chloropromazine or amiloride. NF-kB pathway activation was evaluated by western blot.HaCaT cells stimulation with increasing concentrations of NETs (0.01-50ng/ml) for 48h resulted in a modulation of cell proliferation with a maximum increase corresponding to 0.5-1ng/ml stimulation. NETs low concentrations not only increased cell proliferation, but were also able to induce a faster wound closure in an in vitro scratch assay. NETs scaffold, composed by histone proteins and DNA, is recognized by Toll Like Receptor 9 (TLR 9) that, in turn, activates the NF-kB pathway. In fact, NETs induced proliferation was inhibited by chloropromazine (1nM), that blocks chlatrin vesicles formation, and by amiloride (50nM) that inhibits macropinocytosis. Moreover, dexamethasone, an inhibitor of NF-kB, was able to abolish the NETs effect.This study thus demonstrates that low NETs concentrations undergo internalization finally resulting in a quick NF-kB pathway activation and HaCaT cells proliferation increase, suggesting a close relationship between first immune response and wound healing onset.