Neither eosinophils nor neutrophils require ATG5‐dependent autophagy for extracellular DNA trap formation

Abstract

The importance of extracellular traps (ETs) in innate immunity is well established, but the molecular mechanisms responsible for their formation remain unclear and in scientific dispute. ETs have been defined as extracellular DNA scaffolds associated with the granule proteins of eosinophils or neutrophils. They are capable of killing bacteria extracellularly. Based mainly on results with phosphoinositide 3-kinase (PI3K) inhibitors such as 3-methyladenine (3-MA) and wortmannin, which are commonly used to inhibit autophagy, several groups have reported that autophagy is required for neutrophil extracellular trap (NET) formation. We decided to investigate this apparent dependence on autophagy for ET release and generated genetically modified mice that lack, specifically in eosinophils or neutrophils, autophagy-related 5 (Atg5), a gene encoding a protein essential for autophagosome formation. Interestingly, neither eosinophils nor neutrophils from Atg5-deficient mice exhibited abnormalities in ET formation upon physiological activation or exposure to low concentrations of PMA, although we could confirm that human and mouse eosinophils and neutrophils, after pre-treatment with inhibitors of class III PI3K, show a block both in reactive oxygen species (ROS) production and in ET formation. The so-called late autophagy inhibitors bafilomycin A1 and chloroquine, on the other hand, were without effect. These data indicate that ET formation occurs independently of autophagy and that the inhibition of ROS production and ET formation in the presence of 3-MA and wortmannin is probably owing to their additional ability to block the class I PI3Ks, which are involved in signalling cascades initiated by triggers of ET formation.

DNase I improves corneal epithelial and nerve regeneration in diabetic mice

“…Previous studies have confirmed the effects of ROS accumulation in delaying wound healing of corneal epithelium, 8 and attenuation of ROS accumulation serves as an important therapeutic target for the treatment of delayed corneal regeneration in diabetic condition 27,35 . The formation of NETs depends on the ROS produced by NADPH oxidase, which means that oxidative stress is the key to neutrophil NETosis 36‐38 . This present study consistently showed that DNase I application significantly alleviated the generation of ROS during diabetic corneal epithelial repair by suppressing the expression of NADPH oxidase.…”

Untangling “NETosis” from NETs

“…The majority of these reports have used pharmacological approaches to demonstrate that inhibition of the phosphatidylinositol 3‐kinase (PI3K) type III signaling, which is required for autophagosome formation, prevents NET formation, implying a role for autophagy in NET formation . However, genetic deletion of ATG5 is correlated with defective autophagy, but elicited no defect in extracellular trap formation neither in neutrophils nor in eosinophils , suggesting that autophagy is indeed not required for the formation of NETs (Table ).…”

“…But what are the intracellular events leading to mtDNA release and NET formation? It has been demonstrated that ROS acts as a key signaling molecule for NET formation independent of the stimulus and of the type of granulocyte (Table ). The dependency on ROS was demonstrated using both genetic and pharmacological approaches.…”

Oxidative damage of SP-D abolishes control of eosinophil extracellular DNA trap formation

“…Mouse eosinophils were isolated from IL5 tg mice from bone marrow using Abs against CD8 , CD19, CD90.2, and Ly-6G (Miltenyi Biotec, Bergisch Gladbach, Germany) and an EasySep Mouse PE Positive Selection Kit (Stemcell Technologies) to >95% purity, assessed by the Hematocolor Set (Merck Millipore). 25 Mouse studies were performed in Balb/c, C57BL/6 (wild type, WT) and SP-D −/− mice. O 3 and allergen exposure experiments were carried out as described.…”