Neutrophil Extracellular Traps In Sepsis

Background:Neutrophils are the main effector cells of innate immune in sepsis,with three methods for attacking micro-organisms including:phagocytosis,releasing anti-microbials,and generation of neutrophil extracellular traps(NETs)via NETosis Although NETs are the important mean to defense invading micro-organism,excessive NETs can injure the surrounding cells and tissue.Therefore,the regulation of NETs formation or removal of them may benefit for the septic patients.In this study,we explored the early prognostic value of circulating free DNA/NETs in surgical critically ill patients,and investigated effect of NETs on intestine and the effect of glutamine,common immune-enhancing nutrients,on NETs formation in sepsis,providing a choice for the treatment and research of regulating excessive NETs formation in sepsisPart?PrefaceNeutrophils are the main effector cells of innate immune system in the early inflammation response.their anti-microbial mechanisms including:phagocytosis;releasing anti-microbial substances via degranulation and neutrophil extracellular traps(NETs).The activated neutrophils can release NETs to trap the microorganisms from infection site or blood and kill them.However,on the other hand,the exaggerated NETs formation can injury tissues leading to organs dysfunction,promoting various diseases.The treatment targeting NETs may be a kind of effective therapy.Part ?The early prognostic value of circulating free DNA insurgical critically ill patientsObjectives:The aim of this study was to explore the early prognostic value of circulating free DNA in outcome or risk of sepsis of surgical critically ill patients and possibly related factors.Methods:42 patients were enrolled in the present study.The clinical data were recorded and the SOFA scores and APACHE ? scores were accomplished at admission in ICU.Peripheral venous blood was collected and cf-DNA levels were measured by a direct fluorescence assay.ICU mortality was the primary outcome in ROC cure analysis.Based on the best cutoff in it,the prediction of cf-DNA for the risk of sepsis was explored.Results:Out of 42 patients with cf-DNA concentration of 1.113±0.0530 ug/ml,SOFA of 3.738±0.366 and APACHE II of 9.143±0.752,19%(8/42)have died in ICU.There were significant differences between nonsurvivors and survivors in cf-DNA,SOFA,APACHE II and subsequent sepsis.cf-DNA concentrations at admission in ICU were not associated with SOFA(rho=0.247;P=0.115)and APACHE II(rho=0.092;P=0.564).However them were significantly associated with CRP(rho=0.568;P<0.001)?peripheral white blood cell counts(rho=0.393;P=0.010),neutrophil absolute counts(rho=0.443;P=0.003),and coagulation including aPTT(rho=0.380;P=0.013),PT(rho=0.519;P<0.001),INR(rho=0.518;P=0.001),FIB(rho=0.393;P=0.010),AT?(rho=-0.343;P=0.026).By ROC analysis,cf-DNA concentrations had good discriminative power for ICU mortality(AUC 0.809,95%CI 0.647?0.970),better than CRP(AUC 0.654,95%CI 0.46?0.849).Meanwhile APACHE ?'s AUC was 0.844(95%CI 0.702?0.986)and SOFA's AUC was 0.789(95%CI 0.595?0.982).The best cutoff threshold of cf-DNA was 1.3292 ug/ml,sensitivity was75%(95%CI 34.9%?96.8%)and specificity was 88.2%(95%CI 72.6%?96.7%).The patients in high cf-DNA group had higher risk of sepsis than those in low cf-DNA group(80.0%vs 37.5%,P=0.019*).Conclusion:The correlation of cf-DNA and the synthetic scoring systems needs further study.But cf-DNA is significantly associated with inflammatory biomarker,especially neutrophils,suggesting that neutrophils or NETs may be the important source of cf-DNA.The cf-DNA is associated with coagulation,and is one independent and effective predictor of ICU patients' outcome.Meanwhile higher cf-DNA levels indicate the higher risk of sepsis.Part ?The intestinal injury of neutrophil extracellular traps during sepsisObjectives:The aim of this study was to investigate whether NETs are capable of promoting the impairment of the gut in a rat model of lipopolysaccharide(LPS)-induced sepsis.Methods:The sepsis model was induced in rats by intraperitoneal injection of LPS(10 mg/kg).All rats were divided into three groups as follows:1)control group;2)LPS group;and 3)LPS t DNase I group.The DNase I solution(10 mg/kg)was injected intravenously to disrupt NETs 30 min after the LPS treatment.The animals were sacrificed at 3 h and 24 h after LPS or saline challenge.The intestinal cell apoptosis was examined by detecting the level of cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling assays.The length and morphology of Villi were assessed histologically through hematoxylin and eosin stain.The levels of tumor necrosis factor-alpha and interleukin-10 in serum and intestine were detected by enzyme-linked immunosorbent assay.Intestinal injury was evaluated with Chiu scoring system.Results:A large number of neutrophils infiltrated were activated to release NETs in the intestine of LPS-induced septic rats.The disruption of NETs reduced the acute systemic inflammatory response and apoptosis of intestinal epithelial cells and alleviated histologic pathogenesis.Removal of NETs provided a beneficial effect on intestinal injury.Conclusions:This study demonstrates that the release of NETs may contribute to the intestinal damage during sepsis.Part ?The effect of glutamine on neutrophil extracellular trapsduring sepsisObjectives:The aim of this study was to explore the effect of glutamine,a common immune-enhancing nutrient,on the formation of neutrophil extracellular traps(NETs).Methods:This study included two parts:in vivo animal experiment and in vitro cell experiment.In animal experiment,sepsis was induced via cecal ligation and puncture(CLP)operation.All mice were randomly divided into three groups:1)Sham group;2)CLP group;3)GLN group.The mice received 0.75 g/kg weight glutamine by intraperitoneal(i.p.)injections after CLP operation.Drawing blood was set at four time-point:6 h,12 h,24 h,48 h.The concentration of glutamine and circulating free DNA(cf-DNA)were detected,and the neutrophil-lymphocyte ratio and reactive oxygen species(ROS)were measured via flow cytometry.In cell experiment,the neutrophils of post-surgery patients were activated under different concentrations of glutamine.The area of nucleus and cell free DNA in supernatant were measured to evaluate the formation of NETsResults:The cf-DNA/NETs values were significantly increased in the CLP group compared to the control groups(12 h:P<0.01;24 h:P<0.01;48 h:P<0.05),with a maximum at 12 hours after CLP.Compared with the CLP group,the cf-DNA/NETs values were significantly decreased in the GLN group at 24 hours after CLP(24 h:P<0.01),and were higher than that in the control group(12h:P<0.05;48 h:P<0.05).However the administration of glutamine could not obviously change the ROS levels in peripheral blood neutrophils of septic mice.The neutrophils of post-surgery patients could be activated to release NETs via a process named NETosis with the decondensed nucleus and the citrullination of histones.The neutrophils could still release NETs in the absence of glutamine.The low concentration of glutamine could promote the release of them,but higher concentration of glutamine inhibited this process.Conclusion:The administration of glutamine can reduce the cf-DNA/NETs levels in septic mice.Glutamine is not necessary for the release of NETs,however,different concentrations of glutamine can regulate the formation of NETs.