Tumor Necrosis Factor-a-induced Protein 8-like 2 MRNA Level In Patients With Acute Ischemic Stroke

Part ?:Expression of immunosuppressive molecule TIPE2 in patients with acute ischemic strokeBackgroundAcute ischemic stroke is a multiple complex condition due to an abrupt loss of arterial blood to the brain which will result in the rapid death of the brain tissue.Stroke associated immunity and inflammation are considered to play critical roles in all the stages of disease progression,including acute event of stroke and long term recovery after stroke.When ischemic stroke happens,the brain injury would be initiated by hypoxia inducible factor-la and Notch intracellular domain,which can lead to the production of pro-inflammatory cytokines and the activation of apoptotic signaling pathways.The first responder to brain injury is microglia/macrophage which is an essential modulator of immunologic responses after ischemic stroke.However,the exact mechanism for orchestrating the modulation of immunological response post ischemic stroke has not been well demonstrated.Tumor necrosis factor-a-induced protein 8-like 2(TIPE2)is a recently identified negative modulator of inflammation in maintain immune homeostasis.TIPE2 is highly expressed in resting macrophages and regulates the activation of the NF-?B and activator protein(AP)-1 signaling pathways in innate and adaptive immune response.In the mice model of ischemic stroke,the genetic ablation of tipe2 gene might contribute to more infiltration of macrophages/microglia,neutrophils and lymphocytes in the ischemic hemisphere,and increase the infarction volume of infarction and neurological dysfunction.AimsTo investigate the expression of TIPE2 mRNA and related inflammatory factors in peripheral mononuclear cells of patients with ischemic stroke,and the correlation between TIPE2 mRNA and clinical indicators.MethodsA total of 265 consecutive patients with newly diagnosed as acute ischemic stroke were collected in the Department of Neurology,Jinan Central Hospital affiliated to Shandong University from November 2015 to June 2016.Finally,a total of 83 patients were excluded and 182 patients have been included.Generally,the baseline demographic characteristics were collected as the following The baseline demographic characteristics and relevant risk factors were obtained from medical records.age,sex,,body mass index(BMI),NIHSS score and lesion volume,General laboratory variables.The relative expression of TIPE2 mRNA level in PBMCs was determined by real-time PCR.The expression levels of factor TNF-?,interferon-y,AP-1,NF-?B,IL-6,IL-10,IL-1? mRNA was determined by real-time PCR.The categorical variables were expressed as a percentage(%),the continuous variables were expressed as the median and interquartile(IQR),and the Mann-Whitney U test and the chi-square test were used for comparison between the two groups.Analysis of variance was used to compare the gene expression of AIS patients at different time periods.SNK t test was used to compare between groups.Pearson correlation analysis was used to evaluate the correlation coefficient between TIPE2 mRNA level and various clinical indicators and related factors.The clustering analysis was used to evaluate the similarity of TIPE2 and its related factors.A full-variable two-class logistic regression model was used to analyze the odds ratio(ORd ratio,OR)and 95%confidence interval(CI)of the independent variables to the patient's death outcome.Results1.Descriptive Characteristics of Patients with acute ischemic stroke and healthy controlsThe levels of BMI,HsCRP,GFR,FBG,TG,TC,LDL,and HCY were significantly higher than those in healthy controls(P<0.05,respectively).In the AIS patients,majority of patients were punctured for the determination of TIPE2 mRNA and associated cytokines mRNA levels after the onset at 12-18 hours(n=48,26.37%),followed by 18-24 hours(n=45,24.73%),6-12 hours(n=45,24.73%),more than 24 hours(n=38,20.88%),and less than 6 hours(n=6,3.3%).2.Comparison of TIPE2 and associated cytokines mRNA levels in patients with acute ischemic stroke and healthy controlsThe median relative expression of TIPE2 mRNA in patients with acute ischemic stroke was 4.75 with IQR(3.69-6.38),which was significantly higher that than in healthy controls(2.22,IQR:1.30-4,24;P<0.001).Furthermore,we have determined the relative expression of TIPE2 associated cytokines,including IL-1?,IL-10,IL-6,NF-??,AP-1,IFN-y and TNF-a.In patients with acute ischemic stroke,the relative mRNA levels of TNF-?,AP-1,IFN-? and NF-?? were significantly elevated compared with those in healthy controls.However,there were no significant differences of IL-1? IL-10 and IL-6 in patients with acute ischemic stroke and healthy controls.3.Dynamic profiles of TIPE2 and its associated cytokine mRNA levels in AIS patients with different time stagesThe TIPE2 mRNA level in patients with>24 hours was significant higher than that in patients with<6hours and 6-12 hours(P<005,respectively).In addition,patients with 12-18 hours have significant higher level of TIPE2 mRNA compared with that in patients with 6-12 hours(P<0.05).However,we did not found significant differences of IL-1?,IL-10,IL-6.NF-??,AP-1,IFN-y and TNF-a mRNA levels in AIS patients with different time stages.4.Associations of TIPE2 mRNA levels with laboratory variables in patients with acute ischemic strokeThe median value of lesion volumes was 1.50(0.34-20.25)mL in patients with TIPE2 mRNA<4.75,which was significantly higher than that in patients with TIPE2>=4.75.The median value of NIHSS was 5.00(2.50-11.50)in patients with TIPE2 mRNA<4.75,which was significantly higher than that in patients with TIPE2>=4.75.Pearson correlation analysis was performed and there was significantly negative correlations with TIPE2 mRNA and lesion volumes.5.Associations of TIPE2 mRNA levels with TIPE2 associated cytokines in patients with acute ischemic strokeThe median values of TNF-?,AP-1,IFN-? and NF-?? in patients with TIPE2 mRNA<4.75 were significant higher than that in patients with TIPE2 mRNA>=4.75.Pearson correlation analysis demonstrated that TIPE2 mRNA level was significantly negatively associated with TNF-?,AP-1,IFN-?and NF-??,but significantly positively associated with IL-6 and IL-10.However,there were no significant associations between TIPE2 mRNA and IL-1?.6.TIPE2 and its associated cytokines mRNA levels in survival and nonsurvivals in patients with acute ischemic strokeThe mortality rate of AIS patients with TIPE2 mRNA<4.75(31.87%)was significantly higher than that of AIS patients with TIPE2 mRNA>=4.75(4.40%,P<0.001).violin plot showed that the median of TIPE2 mRNA in survivals was significantly higher than that in nonsurvivals,as well as the same trend for IL-10 in bean plot of Figure 4B.However,the medians of TNF-?,AP-1,IFN-? and NF-??mRNA levels in survivals were significantly lower than that in nonsurvivals.TIPE2 mRNA showed the greatest OR in all the ORs for IL-1?,IL-10,IL-6,NF-??,AP-1,IFN-? and TNF-?,suggesting that TIPE2 mRNA might be a potential biomarker for the mortality of acute ischemic stroke.ConclusionThe expression of TIPE2 mRNA in peripheral blood mononuclear cells of patients with acute ischemic stroke was significantly higher than that of healthy controls.The expression of TIPE2 mRNA was negatively correlated with NIHSS score and infarct size.TIPE2 was negative in patients with acute ischemic stroke.The phase regulates the inflammatory damage of TNF-?.TIPE2 mRNA in peripheral blood mononuclear cells may be a potential marker for clinical prognosis of acute ischemic stroke.Part ?:Peripheral Tumor Necrosis Factor-a-induced Protein 8-like 2 mRNA Level for Predicting 3-month Mortality ofPatients with Acute Ischemic StrokeBackground:Ischemic stroke is a disease with high incidence,high disability rate,high mortality,and high recurrence rate.It is characterized by disruption of cerebral blood flow due to thrombosis or embolism of the cerebral arteries.There are 2.5 million new stroke cases per year in China,and 64.5%are various types of ischemic stroke.Accurate and effective blood biomarkers are necessary to predict the development and prognosis of acute ischemic stroke.Immunity and inflammation play an important role in the development of atherosclerosis,plaque rupture,platelet aggregation,and intravascular thrombosis,and have been shown to be involved in the development of stroke.Therefore,novel immunobiomarkers may be of great value in predicting the prognosis of acute ischemic stroke.Previous literature has reported the potential role of immune molecules as biomarkers for the prognosis of acute ischemic stroke.Wu H et al reported that serum low-tailulin levels can predict early functional outcomes in patients with ischemic stroke.Palm F et al.confirmed that the concentration of serum neutrophil markers is related to the severity and etiology of acute ischemic stroke.These results strongly suggest that immune molecules may be an important factor in the prognosis of acute ischemic stroke.Tumor necrosis factor-?-inducible protein 8-like 2(TIPE2)is a novel immuno-negative regulator that plays an important role in maintaining immune homeostasis and inflammatory responses.Peripheral expression of TIPE2 has been shown to be a molecular biomarker for the diagnosis of liver and kidney disease.Studies have shown that TIPE2 may have a protective effect on animal models of ischemic stroke.It is speculated that targeting TIPE2 may be a new therapeutic strategy for stroke therapy.Therefore,we speculate whether TIPE2 mRNA can be used as a biomarker to predict functional prognosis and mortality in acute ischemic stroke.Aims:This study aimed to investigate TIPE2 mRNA in peripheral blood mononuclear cells(PBMCs)for predicting 3-month functional outcomes and mortality of patients with acute ischemic stroke.Methods:This study prospectively included 182 patients who met the diagnostic criteria for acute ischemic stroke.The TIPE2 mRNA expression levels in peripheral blood mononuclear cells were measured at admission.All patients were followed up for three months with a modified Rankin scale(mRS)score as a function.Bad judgment criteria,with survival or death as all-cause deaths.The effects of TIPE2 mRNA and other routinely used biomarkers on the mortality and functional outcome were calculated using Cox proportional hazards regression models.Univariable analysis with a crude model was performed to identify the hazards ratio(HR)and the 95%confidence interval(CI)of each confounder.Multivariate analysis was performed to estimate the non-adjusted and adjusted HR and 95%CI of functional outcome and mortality for TIPE2 increase per unit,standard deviation(SD),and TIPE2 quartiles(with the lowest TIPE2 quartile as reference).In model ?,the adjusted confounders were set on the basis of the outcomes of interest or a change in effect estimate of more than 10%,as well as the P value less than 0.1 for the regression coefficient of each confounder in Model ?.The confounder "age" was adjusted in both model ? and Model ?.Tests for linear trends in HRs across TIPE2 quartiles were performed with the median within each quartile used as the predictor.The diagnostic accuracy was assessed by the receiver operating characteristic(ROC)curve,and reported by the area under the curve(AUC),as well as sensitivity,specificity,positive likelihood ratio(LR+),negative likelihood ratio(LR-),positive predictive value(PPV)and negative predictive value(NPV).The calibration of biomarker has been represented as calibration curve,and net reclassification improvement(NRI)and integrated discrimination improvement(IDI)have also been performed.The survival probability for patients stratified by TIPE2 quartiles was calculated by using Kaplan-Meier survival curves.The time dependent tree analysis was also pruned using recursive partition for decision making in the clinic.Research Result:1.Descriptive Characteristics of Patients with acute ischemic strokeThere were 90(49.5%)patients with male,97(53.3%)patients with family history of acute ischemic stroke,102(56.0%)patients with cigarette smoking habit,106(58.2%)patients with alcohol drinking,120(65.9%)patients with hypertension,57(31.5%)patients with diabetes mellitus,48(26.4%)patients with coronary heart disease,and 16(8.8%)patients with atrial fibrillation.According to the criteria of OSCP,the major clinical stroke syndromes types was TACI(110,60.4%),followed by POCI(48,26.4%),PACI(18,9.9%)and LACI(6,3.3%).At the end of the 3-month follow-up,there were 55(30.2%)patients with an unfavorable outcome and 33(18.1%)patients had died.2.Associations of TIPE2 mRNA level with clinical parameters in patients with acute ischemic strokeThe relative TIPE2 mRNA level was negatively associated with NIHSS score(r=-0.154,P=0.039),lesion volumes(r=-0.228,P=0.002),and TG(r=-0.204,P=0.042).However,there was no significant relationship between the relative TIPE2 mRNA levels with HSCRP(r=-0.078,P=0.295).There were no significant associations of TIPE2 mRNA levels with age,sex,BMI,family history of acute ischemic stroke,cigarette smoking habit,alcohol drinking habit,hypertension,coronary heart disease,atrial fibrillation,eGFR,SBP,DBP,TC,LDH,D-dimer,or OSCP score(all P>0.05,respectively).3.TIPE2 mRNA level and 3-month mortalityMultivariate analysis showed the adjusted HR of the highest quartile(Q4)was 0.024(95%CI 0.003-0.196)for mortality in Model ? and 0.018(95%CI 0.002-0.169)in Model ? compared with the reference category(QI).Each per SD increase of TIPE2 mRNA level was associated with a 76.0%(HR 0.240,95%CI 0.126-0.455)decreased in Model ?,as well as a 79.9%(HR 0.201,95%CI 0.093-0.432)decreased risk in Model ? for mortality.4.TIPE 2 mRNA as biomarker for predicting 3-month mortalityAUC for TIPE2 mRNA for mortality was 0.810(95%CI 0.733-0.886),with the optional cut-off value 5.175 as well as 51.0%for specificity,97.0%for sensitivity,1.979 for LR+,0.059 for LR-,0.305 for PPV and 0.987 for NPV.In addition,TIPE2 mRNA was superior to Hs-CRP(AUC 0.739,95%CI 0.633-0.845,P<0.001)and TG(AUC 0.702,95%CI 0.601-0.804,P<0.001),but significantly inferior to NIHSS score(AUC 0.882,95%CI 0.833-0.9325,P<0.001).To assess the combined influence of TIPE2 mRNA and NIHSS score on mortality,a new variable predicting probability was established according to an equation obtained by binary logistic regression:TIPE2 plus NIHSS(3-month mortality)= 0.38532-0.98885*TIPE2 +0.24366*NIHSS.The AUC of NIHSS plus TIPE2(0.925,95%CI 0.874-0.976)was significantly greater(P=0.04)than NIHSS score(AUC=0.882,95%CI 0.833-0.932).Calibration curve showed good performance of NIHSS plus TIPE2 in predicting 3-monthly mortality.NRI and IDI of TIPE2 plus NIHSS were also significantly better than NIHSS or TIPE2 alone.Therefore,time to death was analysed by Kaplan-Meier curve according to the four quartiles of TIPE2 mRNA.Patients with TIPE2 mRNA levels in the upper two quartiles(TIPE2 mRNA>6.382 and TIPE mRNA between 4.755 and 6.382)had a lower risk of death compared to patients with TIPE2 mRNA levels in the lower two quartiles(TIPE2 mRNA<3.69 and TIPE2 mRNA between 3.69 and 6.382;log-rank=30.1242,P<0.001).Furthermore,the time dependent tree analysis was also pruned using recursive partitioning for decision making in the clinic.At the end of the 3-month follow up,only 2 patients had died(1.8%)in the group with NIHSS<5.5(n=109).When the NIHSS score was greater than 5.5,only 1 patient had died(4.8%)among the patient with TIPE2 mRNA>=5.2(n=21),but 30 patients died(57.5%)in the patients with TIPE2 mRNA less than 5.2(n=52).Therefore,AIS patients with NIHSS>=5.5 and TIPE2 mRNA<5.2 had rather high 3-month mortality rate and should be closely monitored in the clinic.5.TIPE2 mRNA level and 3-month functional outcome Multivariate analysis showed the adjusted HR of the highest quartile(Q4)was 0.194(95%CI 0.074-0.509)for unfavorable functional outcome in Model ? and 0.105(95%CI 0.035-0.320)in Model ? compared with the reference category(Ql).Each per SD increase of TIPE2 mRNA level was associated with a 50.0%(HR 0.500,95%CI 0.342-0.733)decreased risk in Model ?,as well as a 63.1%(HR 0.369,95%CI 0.241-0.563)decreased risk in Model ? for the unfavorable functional outcome.6.TIPE 2 mRNA as biomarker for predicting 3-month unfavorable outcomeAUC of TIPE2 mRNA for 3-month unfavorable functional outcome was 0.740(95%CI 0.662-0.818),with the optional cut-off value 4.980 as well as 57.5%for specificity,83.6%for sensitivity,1.967 for LR+,0.284 for LR-,0.460 for PPV and 0.890 for NPV.TIPE2 mRNA was similar to Hs-CRP(AUC 0:750,95%CI 0.662-0.838,P>0.05)and TG(AUC 0.739,95%CI 0.657-0.821,P>0.05),but significantly inferior to NIHSS score(AUC 0.830,95%CI 0.768-0.891,P<0.01).To assess the combined influence of TIPE2 mRNA and NIHSS score on the prediction of 3-month functional outcome,a new variable predicting probability was established according to an equation obtained by binary logistic regression:TIPE2 plus NIHSS(functional outcome)= 0.23926-0.50198*TIPE2 +0.17408*NIHSS.TIPE2 mRNA did not improve(P>0.05)the AUC of the NIHSS score(AUC for the combined model =0.841,95%CI 0.774-0.908,).However,calibration curve showed good performance of NIHSS plus TIPE2 in predicting 3-monthly unfavorable functional outcome.NRI and IDI of TIPE2 plus NIHSS were also significantly better than NIHSS or TIPE2 alone.Conclusion:Our present study first demonstrated that TIPE2 mRNA was an independent risk factor for functional outcome and mortality in Chinese patients with acute ischemic stroke.Furthermore,we proposed that acute ischemic stroke patients with NIHSS>=5.5 and TIPE2 mRNA<5.2 had rather high 3-month mortality and should be closely monitored in the clinic.Large and multicenter cohorts are needed to test and validate the diagnostic value of TIPE2 in predicting the outcome of acute ischemic stroke.