Abstract
Neutrophils release neutrophil extracellular traps (NETs), via NETosis, as a defense mechanism against pathogens. Neutrophils can release NETs spontaneously; however, the mechanisms underlying spontaneous NETosis remain unclear. Neutrophils isolated from healthy donors were tested for NET formation and autophagy at 1, 6, 12, and 24 h after incubation. Autophagy response was evaluated in response to various autophagy inducers and inhibitors. The relationship between autophagy and NETosis was detected in vivo using an ovalbumin-induced mouse model of asthma. We found that the increase in the proportion of spontaneous NETosis was time-dependent. The number of autophagy-positive cells also increased over time and LC3B protein played an integral role in NET formation. Trehalose (an inducer of mTOR-independent autophagy) treatment significantly increased NET formation, whereas rapamycin (an mTOR-dependent autophagy inducer) did not increase NET release by neutrophils. Compared with the control group, 3-methyladenine (an autophagy sequestration inhibitor) and hydroxychloroquine sulfate (autophagosome-lysosome fusion inhibitor) treatments significantly reduced the percentage of NET-positive cells. In vivo studies on ovalbumin-induced asthma lung sections revealed NETs and LC3B and citH3 proteins were found to co-localize with DNA. Our findings suggest that autophagy plays a crucial role in aging-related spontaneous NETosis.
Highlights
During NETosis[11,17,18]
Given the short half-life of neutrophils[24], we hypothesized that the spontaneous formation of neutrophil extracellular traps (NETs) was time-dependent
To investigate various time-points and efficiency of spontaneous NETosis, we tested the proportion of spontaneous NET formation after 1, 6, 12, and 24 h of incubation
Summary
During NETosis[11,17,18]. Upon phorbol 12-myristate 13-acetate and lipopolysaccharide stimulation, autophagosomes containing cytosolic components were observed in neutrophils by transmission electron m icroscopy[18,19]. The association between NETosis and autophagy was confirmed using various autophagy inducers and inhibitors, which revealed that autophagy inducers improve the release of NETs in response to various stimulations, whereas autophagy inhibitors abrogate tis e ffect[18,19,20]. We hypothesized that autophagy was involved in the mechanism of spontaneous NETosis. We explored the mechanism of spontaneous NETosis, thereby adding to the existing knowledge available on this phenomenon
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