The Mechanism Of Coronary Artery Lesions Induced By Neutrophil Extracellular Traps In Kawasaki Disease

Objective:To investigate the changes of Neutrophil Extracellular Traps in children with Kawasaki disease（KD）and it roles in the possible mechanism of coronary artery lesion.Methods:Thirty-eight children with KD were enrolled in the present study during admitted in Shenzhen Children’s Hospital from May 2020 to December 2020,which fulfilled with the diagnosis guidelines of Kawasaki disease proposed by American Heart Association（AHA）in 2017 as twelve age-matched health children as control group（Ctrl group）.Peripheral blood was collected for examination（blooding before or after IVIG treatment）.The patients after IVIG treatment were included in the IVIG treatment group(KD^IVIG group).Refer to the report of doppler ultrasound,divided acute KD group into coronary artery injury subgroup（CAL group）and no coronary injury subgroup（CAL group）.（1）Flow cytometry was used to detect the proportion of neutrophils,the number of NETs,the concentration of ROS and the expression of PAD4 and LC3II in peripheral blood neutrophils.（2）Quantitative real-time PCR was used to evaluate levels of IL-6、IP-10、TNF-α、My D88 m RNA in neutrophils.（3）Plasma concentrations of Histone H3and H4 were measured by enzyme-linked immunosorbent assay.Results:（1）The proportion of neutrophils in peripheral blood of children with acute KD was significantly increased and accompanied by the number of NETs increased（P<0.05）,as the proportion of neutrophils and the number of NETs in CAL group were higher than those in NCAL group（P<0.05）.The proportion of neutrophils decreased significantly after IVIG treatment（P<0.05）.（2）The concentration of ROS and the expression of PAD4 and LC3II,the upstream regulatory factors of NETs formation by peripheral blood neutrophils in children with acute KD,were significantly higher than those in control group（P<0.05）.The concentration of ROS in neutrophils from patients treated with IVIG was lower than that in patient with acute（P<0.05）.（3）Transcription levels of IL-6,My D88,IP-10 and TNF-αm RNA in neutrophils from children with acute KD were significantly higher than those in control group（P<0 05）.The transcription levels of IP-10 and TNF-αm RNA in patients treated with IVIG were decreased（P<0 05）.The level of IP-10 m RNA transcription in CAL group was significantly higher than that in NCAL group（P<0.05）.（4）Compared with control group,plasma concentrations of histone H3 and H4 in children with acute KD increased significantly（P<0.05）,as plasma concentrations of H3 and H4 in CAL group were much higher than those in NCAL group（P<0.05）.There was no significant difference in the concentration of histone H3 and H4 between patients treated with IVIG and patient with acute（P>0.05）.Conclusion:（1）The activation of neutrophils and the formation of NETs were enhanced in the acute phase of KD,especially in patients with coronary artery lesions.Furthermore,the production of ROS and PAD4 in the upstream of NETs increased,as the concentration of NETs structure-related histone H3 and H4 in plasma were elevated during acute KD,which suggested that NETs was involved in the process of vascular inflammatory injury in KD.（2）Transcription levels of My D88,IL-6,IP-10 and TNF-αgene,which are related to the activation of NETs as regulatory molecules of p DCs,increased during acute phase of KD,suggesting that NETs may further activate T cells and B cells and cause vascular injury via activating p DCs in KD.（3）The related markers of NETs（such as cf DNA,PAD4,histone,etc.）and their upstream and downstream molecules（such as ROS,autophagy,inflammatory factors,etc.）can be used as biomarkers for early evaluation of CAL,and therapeutic targets.