Association Of Circulatory Cell-free DNA With Immune Inflammatory Mechanism And Prognosis In Children With Sepsis

ObjectiveSepsis is life-threatening organ dysfunction caused by a dysregulated host response to invasive infection,characteristic of excessive systemic inflammatory response syndrome（SIRS）at early stage and a long period of immune suppression with imbalance of SIRS and compensatory anti-inflammatory response syndrome（CARS）and decreased expression of HLA-DR in the late period.The catastrophic pathophysiology of sepsis included SIRS,SEPSIS,SEVERE SEPSIS/SEPTIC,MODS,and DEATH.Given a highly sensitive and specific biomarker to timely reflect the pathophysiology,the prognosis of sepsis certainly would be improved.Nowadays,several biomarkers such as IL6,IL10,TNFα,HMGB1,CRP,PCT,and HLA-DR,have been proved to be unable to accurately predict injury risk relate to MODS/DEATH.Circulatory cell-free DNA（cfDNA）was likely released by several inflammatory cells and necrosis or apoptotic cells.Given that cell death was a common occurrence during sepsis,cfDNA has recently been explored as a prognostic biomarker for sepsis.But,the association between cfDNA and inflammatory immune markers and predictive value of cfDNA in septic children remain to be verified.The first aim of present study was to explore the association of dynamics of cfDNA among inflammatory biomarkers,acute phase proteins,and immune response and prognostic value in different stage of sepsis by establishing a mice model of sepsis-induced MODS.Secondly,a prospective clinical study was designed to prove the practical availability of cfDNA in children with sepsis.Methods1.Experimental study:Twenty of 4-year old male M1-dominant C57BL/6 mice and M2-dominant BALB/c mice were randomly divided into experimental groups（n=10）and control groups（n=10）,respectively.The experimental groups of two mouse strains were treated with an intraperitoneal injections of E.coli to establish the model of sepsis-induced MODS.The levels of murine sepsis score（MSS）,TNFa,IL10,hepatic and renal function,myocardial enzyme,arterial lactate,arterial blood gas analysis,and organ pathologic score were measured and compared between two groups to prove availability of the model.After success of establishing the model,the levels of cfDNA,TNFa,IL10,monocyte subsets(CD115+Ly6C^high and CD115+Ly6C^low,Ly6CH and Ly6CL),Ly6CH/L,organ functions,arterial lactate,MSS,organ dysfunctional score（ODS）were compared,and explored the relation to each other and predictive effect on outcomes at before injection（0d）and 0.25d（6h）,0.5d（12h）,1d（24h）,2d,3d,4d,5d,6d,7d after injections.2.Clinical study:Three hundreds children aged 3.25±2.9 years old including 180 male cases who met the diagnosis of sepsis were included in a clinical prospective observational study.All of patients treated with the standard therapy of sepsis were divided into MODS group vs.Non-MODS group and survival group vs.death group in term of MODS/death occurred or not at 24h and 96h after admission.The levels of cfDNA,HLA-DR and biomarkers（IL6,TNFa,IL10,HMGB1,CRP,PCT）were detected and compared between two groups at 24h and 96h,respectively.Association of cfDNA with the biomarkers was analyzed,and prognostic value of cfDNA and the biomarkers for outcome（28d-mortality）was compared at 24h and 96h.Results1.Experimental study:（1）The levels of MSS,organ pathologic score,TNFa,IL10,ALT,AST,cTnI,BUN,Scr,Lac,PaCO2,Ly6CH and Ly6CH/L in experimental groups of both strains were significant higher than that in control groups（P<0.05）,and PH,PaO2,Ly6CL were lower in experimental groups（P<0.05）.Comparison between two strains,the levels of TNFα,IL10,ALT,AST,cTnI,BUN,Scr,Lac,PaCO₂,Ly6CH and Ly6CH/L in M1-dominant C57BL/6 mice were higher than that in M2-dominant BLAB/c mice（P<0.05）,and PH,PaO₂,Ly6CL were lower in M1-dominant C57BL/6 mice（P<0.05）.（2）The levels of cfDNA,TNFa,Ly6CH and Ly6CH/L in both strains gradually increased after injections,and reached peak values at 3th days then decreased gradually at the end of observation.The levels of biomarkers mentioned above in C57BL/6 mice from 0.25^th days were higher than that in BALB/c mice,and IL10,Ly6CL were lower in C57BL/6 mice（P<0.05）.There were positive correlation of cfDNA and TNFain C57BL/6 mice（r=0.773,P=0.00）,cfDNA and IL10 in BALB/c mice（r=0.799,P=0.00）with high correlated one-phase association in both strains（R²=0.65,0.639）.Analysis of correlation between cfDNA and M1/M2 response showed significant positive correlation（r=0.899,P=0.00）with high correlated one-phase association in both strains（R²=0.833）.（3）The levels of ALT,AST,cTnI,BUN,Scr,Lac,PaCO₂,ODS and MSS in both strains gradually increased after injections,and reached peak values at 3th days then decreased gradually at the end of observation.The levels of biomarkers mentioned above in C57BL/6 mice from 0.25^th days were higher than that in BALB/c mice,and PH,PaO₂ were lower in C57BL/6 mice（P<0.05）.There were positive correlation of cfDNA and MSS,ODS in both stains（r=0.786,0.799,P=0.00）with high correlated one-phase association in both strains（R²=0.618,0.739）.（4）Except for IL10,all of cfDNA,TNFa,Ly6CH/L,MSS,ODS could predict 7d-mortanlity at 0.25d after injections in both two strains（P<0.05）.The area of under curve（AUC）of receiver operation character（ROC）curve of cfDNA（0.95）and Ly6CH/L（0.92）to 7d-mortanlity were highest.At 0.5d and Id after injections,all of cfDNA,IL10,Ly6CH/L and ODS could predict 7d-mortanlity except TNFaand MSS（P<0.05）.The AUC of ROC of cfDNA（0.95）and Ly6CH/L（0.92）to 7d-mortanlity were highest,too.There were positive correlation between cfDNA and TNFa,Ly6CH/L,MSS,ODS at 0.25d and 0.5d,and negative correlation with IL10.Ly6CH/L has the most relevance to cfDNA both at 0.25d and 1d（r=0.93.0.91,R²=0.904₀.81,P=0.00）.At 1d after injection,positive correlation was found between cfDNA and IL10,Ly6CH/L,ODS,MSS and negative correlation with TNFα.Ly6CH/L also has the most relevance（r=0.91,R²=0.822,P=0.00）.（5）The AUC of ROC of cfDNA at 0.25d after injection to 7d-mortanlity was 0.934,the cut-off value of cfDNA was 505.5ng/ml.Kaplan-Meier survival curve showed that there has significant lower survival time in high cfDNA group（≥505.5ng/ml）and M1-dominant C57BL/6 mice than that in low cfDNA group（<505.5ng/ml）and M2-dominant BALB/c mice.2.Clinical study（1）The levels of cfDNA,HLA-DR,IL6,TNFa,CRP,and PCT were significant higher in MODS group than that in Non-MODS group（P<0.05）,and there were no difference in HMGB1 and IL10（P>0.05）at 24h after standard treatment.At 96h,the levels of cfDNA,HLA-DR,HMGB1,and IL10 were significant higher in MODS group than that in Non-MODS group（P<0.05）,and there were no difference in IL6 and TNFa（P>0.05）.（2）A linear relation was found between 24h-cfDNA and HLA-DR,IL6,TNFα,respectively.Then between 96h-cfDNA and HLA-DR,HMGB1,and IL-10 showed a linear relation,too.（3）The cfDNA as a predictive marker for outcome（28d-mortanlity）at different stage of sepsis was equal to pediatric risk of mortality score III（PRISM-III）,and significant higher than pediatric clinical illness score（PCIS）and other inflammatory factors.The higher risk of MODS or death has occurred both in high 24h-and 96h-cfDNA groups than that in low groups.High cfDNA groups have lower survival time.（4）Kaplan-Meier survival curve revealed that there has lower survival time in high cfDNA group and decreased expression of HLA-DR group at 24h and 96h after standard treatment.ConclusionThe conclusions of experimental study and clinical study are consistent:1.The main cause of MODS/DEATH at early stage of sepsis is imbalance of SIRS/CARS characterized by excessive hyperinflammatory response and dysfunction of innate immunity;then,gradually increased anti-inflammatory response and sequential immune paralysis for a long period lead to MODS/DEATH at late stage.2.cfDNA is a marker for the damaged tissue during sepsis and could reflect the degree of imbalance of SIRS/CARS and disorders of immune response.3.cfDNA can be a valuable and easily available prognostic factor for survival status and outcome.The present study suggested that cfDNA is a good indicator for evaluating prognosis of pediatric sepsis in clinical practice.