MiR‐1696/GPx3 axis is involved in oxidative stress mediated neutrophil extracellular traps inhibition in chicken neutrophils
Zijiang Yang, Shengchen Wang, Kai Yin, Qiaojian Zhang, Shu Li
Abstract
Abstract As an important immune mechanism of neutrophils, the release of Web‐like chromatin structures known as neutrophil extracellular traps (NETs) can rapidly locate and capture invading pathogens, which has received sustained attention. There are still some fundamental questions surrounding established studies on the mechanism of balance between reactive oxygen species (ROS) dependent release and neutrophil antioxidant response. Glutathione peroxidase 3 (GPx3) is an important antioxidant protein and has been identified can regulate the immune response. However, the effect of GPx3 on the NETs formation and microRNA in this process remain poorly understood. In the present study, we used chicken peripheral blood neutrophils treated with Phorbol‐12‐myristate‐13‐acetate (PMA) for 3 h as NETs formation model. The result of morphological observation showed that GPx3 inactivation compromised the release of NETs. Further analysis revealed that knockdown of GPx3 significantly disturbed oxidative balance by inhibiting antioxidant enzymes activity and increasing H 2 O 2 content. Quantitative analysis of NETs‐related genes found that the phosphorylation level of mitogen‐activated protein kinase (MAPK) pathway genes (ERK, JNK, and p38) and expression of phosphoinositide‐3‐kinase (PI3K)/AKT pathway genes (PI3K and AKT) were suppressed with the downregulation of GPx3. Meanwhile, we identified that miR‐1696 can target GPx3 expression by using dual luciferase reporter system. Additionally, overexpression of miR‐1696 can not only inhibit the formation of NETs by restraining the expression of GPx3, interfering with the generation of ROS and activation of the MAPK and PI3K/AKT pathways, but also reducing the release of PMA‐induced NETs promoted by overexpression of GPx3. These results provide evidence that miR‐1696 targeted GPx3 activities in neutrophils could be used to regulate the NETs formation stimulated by PMA.