Investigation And Assessment Of Neutrophil Dysfunction At The Early Stage Of Severe Burn Injury

Background and objective:Burn injury is a common thermal trauma in the community life,the severity and mortality rate of burn injury grow with increasing burn area,depth,age and smoke inhalation.Severe burn injuries,especially extensive burns,can result in massive fluid loss during the shock stage and a more complex interplay of pro-and anti-inflammatory signals,leaving burn-injured patients in a state of immune system dysregulation.At present,some researches on the relationship between immune system and the occurrence and development of sepsis in severe burn-injured patients have been reported.Nevertheless,the role of the neutrophils,one of the most significant innate immune cell,in the burn shock stage has rarely been explored.The aim of this study was to comprehensively investigate and assess the function of neutrophils during shock stage in patients with severe extensive burn injury,and to explore the relationship between the changes of neutrophil function and pathophysiological reaction during the burn shock stage.Methods:In vivo experiments,12 male C57BL/6 mice were randomly divided into control group（n=3）,sham operation group（Sham group,n=3）,extensive burn model group（Burn group,n=3）and recombinant heparin binding protein injection group（r HBP group,n=3）.The Texas Red Dextran was injected into the tail vein as a blood tracer,and neutrophils were labeled with biotin anti-mouse Ly-6G and i Fluor^TM647-streptavidin conjugate.Laser scanning confocal microscopy（LSCM）was used for in vivo observation of the abdominal vessels and neutrophils in mice after modeling.In vitro experiments,6 male C57BL/6 mice were randomly divided into control group（n=3）and extensive burn model group（n=3）.The peripheral blood neutrophils of mice were separated by magnetic beads,and the nucleus was stained with dapi.The morphology of neutrophil nucleus was observed under LSCM.Meanwhile,the peripheral blood from 20 healthy adult volunteers and 18 extensive burn-injured patients during shock stage were collected.Plasma samples were obtained by centrifugation and preserved carefully.Neutrophils were separated using magnetic beads and cell karyotypes were observed.The chemotactic function of neutrophils was assessed and analyzed by under agarose chemotaxis model and cell chemotactic analysis platform（CCAP）.The function of phagocytosis,level of degranulation,ability of respiratory oxygen burst,apoptosis rate of 24-hour and the expression of P2X1 receptor on the surface of cells were detected by flow cytometry.The state of degranulation and the generation of neutrophil extracellular trap（NET）were detected by LSCM.Enzyme-linked immunosorbent assay（ELISA）detecting kits were used to detect the concentration of antibacterial proteins（myeloperoxidase [MPO],heparin-binding protein [HBP],matrix metalloproteinase-9 [MMP-9] and neutrophil elastase [NE]）in plasma of volunteers and patients.The laboratory data and clinical information of patients were summarized and analyzed statistically.Results:The results of in vivo experiments indicated that compared with Control group and Sham group,there were obvious blood viscosity and vascular leakage in r HBP group and Burn group during shock stage--a large amount of plasma leaked from blood vessels to tissue space.Compared with Control group,the number of neutrophils in Burn group during shock stage was significantly increased,most of the increased neutrophils adhered to the surface of vascular endothelial cells,and the chemotactic function decreased significantly.The results of in vitro experiments indicated that the nuclear images of neutrophils changed in both burn-injured patients and burn model mice,showing a typical nuclear shift to the left.The number and percentage of neutrophils in peripheral blood of severely burn-injured patients during shock stage were conspicuously increased compared with those of healthy controls,but the chemotactic function was obviously impaired,and there was a significant negative correlation（r=-0.9398,P=0.0002）between 2-hour chemotactic distance and the expression of P2X1 receptor on neutrophil surface.The ability of neutrophil degranulation and the concentration of plasma antibacterial protein（MPO,HBP,MMP-9,NE）increased significantly,and the massive release of HBP caused by degranulation was demonstrated to be related to the loss of body fluid in burn shock stage.What’s more,the phagocytosis of neutrophils,the ability of respiratory oxygen burst and the generation of NETs were decreased.Apoptosis in burn-injured patients was delayed and the proportion of apoptosis was lower than that in healthy volunteers.Conclusions:The functions of neutrophils in peripheral blood of severe burn-injured patients during shock stage were significantly abnormal,including impaired chemotactic ability,decreased phagocytic function and changed bactericidal function.It is revealed that there is a close-knit relationship between the changes of pathophysiological responses in burn shock stage and the dysfunction of immune system.Focusing on the recovery of neutrophil function will help to reduce the fluid loss during shock stage,improve the immune system function and reduce the severity of subsequent infection in burn-injured patients.It provides a new insight and direction for clinical evaluation and treatment of extensive burn-injured patients at the shock stage.