Expression And Clinic Significance Of TIPE2 MRNA In Peripheral Blood Mononuclear Cells From Patients With Chronic Hepatitis B Virus Infection

Part Ⅰ:Expression of TIPE2 mRNA in PBMCs from Patients with Chronic Hepatitis B, Liver Cirrhosis and HepatocellularCarinomaBackgroudHepatitis B virus (HBV) infection is the major cause for the prevalence of viral hepatitis in China. Almost 90% of acute HBV infection will progress into recovery in adult; however, one can develop into chonric hepatitis if the dyfunction of adaptive and innate immune response. Chronic HBV infection could cause liver fibrosis and eventually develop into liver cirrhosis (LC) with effective prevention. Furthermore, HBV is also the leading cause for the development of hepatocellularcarcinoma (HCC). HCC accounts for approximately 70%-80% of all primary liver cancer cases and is characterized by a progressive development and poor prognosis. Recent studies have provided evidence that immune system dysregulation plays an important role in the fate of HBV progression in humanbeing.Tumor necrosis factor-α-induced protein 8-like 2(TIPE2) is a newly identified negative regulatory molecular of immune response, and governs immune homeostasis by negatively regulating signaling through T cell receptors and Toll-like receptors. In recent years, accumulating studies reported that TIPE2might play an important role in the development of chronic inflammatory diseases, autoimmune disorders, stroke, diabetic nephropathy, carcinoma and atherosclerosis. Zhang W et al reported the aberrant expression of TIPE2 in hepatitis B patients and revealed that TIPE2 might regulate HBV specific CD8 (+) T cells function and contribute to the pathogenesis of hepatitis B. In the patients with ACHBLF, we also previously reported the elevation of TIPE2 mRNA and speculated that TIPE2 might negatively regulate cell-mediating immunity to abnormal liver function in a preliminary study. However, there is no data of TIPE2 in the pathogenesis of liver cirrhosis and hepatocellular carcinoma. Therefore, it is essential to make clear the dynamic expression of TIPE2 in the patients with chornic hepatitis B, liver cirrhosis and hepatocellular carcinoma. AimsThis present study was to determine the dynamic expressions and clinic significance of TIPE2 mRNA in peripheral blood mononuclear cells (PBMCs) from patients with chornic HBV infection, including chornic hepatitis B, liver cirrhosis and hepatocellular carcinoma.MethodsThis retrospective and cross-sectional study included a total of 197 patients with chronic HBV infection (97 for chronic hepatitis B,55 for liver cirrhosis patients and 41 for HBV-related hepatocellular carcinoma patients) in the Department of Hepatology, Qilu Hospital of Shandong University from Dec 2013 to May 2014. There were fourteen healthy volunteers as control. We determined the mRNA expression levels of TIPE2 in PBMCs from all enrolled patients and 14 healthy controls using relative quantitative real-time polymerase chain reaction (RT-PCR).Results1. The relative TIPE2 mRNA levels of PBMCs in patients with liver cirrhosis and chronic hepatitis B were significanty higher that that in healthy controls (both P<0.05). There was no signiciant difference of TIPE2 mRNA level in the patients with liver cirrhosis and chornic hepatitisB (P>0.05). Furthermore, the relative TIPE2 mRNA level in patients with hepatocellular carcinoma was significantly lower than that in healthy controls (P<0.01).2. In chronic hepatitis B patients, we demonstrated a significant higher level of TIPE2 mRNA in HBVDNA positive patients compared with HBVDNA negative patients (2.27 [1.38,3.20] vs.1.34 [0.84,2.33], P< 0.05). Spearman analysis showed that TIPE2 mRNA expression in PBMCs was positively correlated with serumALT, AST and PTA(r=0.262, P<0.05; r=0.292, P<0.05;r=0.207, P<0.05).3. The TIPE2 mRNA level in decompensated LC patients wassignificantly higher than that in compensated LC patients (1.36 [0.92,1.74] vs.2.33[1.44,3.18], P< 0.05). TIPE2 expression was positively correlated with TBIL and MELD score in LC patients (r=0.288, P=0.033; r= 0.366, P< 0.01)4. In HBV-related HCC patients, TIPE2 expression was significantly correlated with primary tumor size, venous invasion and TNM staging.Furthermore, TIPE2 expression was negatively correlated with AFP(r=0.312, P<0.05).ConclusionThe relativeTIPE2 mRNA levels of PBMCs in patients with liver cirrhosis and chronic hepatitis B were significanty higher that that in healthy controls; while the relative TIPE2 mRNA level in patients with hepatocellular carcinoma was significantly lower than that in healthy controls. The results suggest that TIPE2 mRNA is associated with liver injury and disesase severity in patients with chornic hepatitis B and liver cirrhosis. However, TIPE2 mRNA is correlated with TNF stage of HCC patients. TIPE2 is involved in the progression of chornic HBV infection.Part II:TIPE2 mRNA in PBMCs is Associated with Disease Progression of Chronic Hepatitis B Virus InfectionBackgroudsHepatitis B virus (HBV) infection is a serious threat to public health. And it is estimated that there are almost more than 350 million HBV carriers worldwide.Immune system is demonstrated to be one of the main elements for the different outcome of chronic HBV infection.Therefore, aberrant immune response in chronic HBV infection might participate in the diseases progression of a spectrum of HBV associated liver diseases, ranging from chronic hepatitis B, and liver failure to liver cirrhosis. It is now recognized that the natural course of chronic hepatits B (CHB) is typically divided into four phases:the immune-tolerant (IT) phase, the immune clearance (IC) phase, low or no-replicative (LR) phase andhepatitis B e antigen (HBeAg)-negative hepatitis (ENH) phase. Understanding the natural history of CHB makes a lot of sense on antiviral treatment. However, the exact function and mechanism of immune regulation in the natural course of chronic HBV infection remains still obscure.Tumor necrosis factor-a-induced protein 8-like 2(TIPE2) is a new member of negative immune regulator, and governs immune homeostasis in both the innate and adaptive immune systems. We and other researchers demonstrated that TIPE2 participate in the pathogenesis of chronic hepatitis B and liver failure. However, there is no data of TIPE2 mRNA expression in different immune phases of chornic hepatitis B.Aims:1. To detect the expression of TIPE2 mRNA in PBMCs and TIPE2 associated cytokines in different immune phasesof chronic hepatitis B patients.2. To determine the expression of hepatic TIPE2 protein in CHB patients.MethodsA total of 205 naive treated patient with chronic hepatitis B and 15 healthy volunteers as control were enrolled in the Department of Hepatology, Qilu Hospital of Shandong University from Dec 2013 to Jan 2015. Of these patients, there were twenty-five CHB patients with a liver biopsy. Additionally, four liver speciments w from healthy liver transplant donors sevred as normal. We determined the mRNA expression levels of TIPE2, IL-6, IL-10, TNF-a, and IFN-y in PBMCs using relative quantitative real-time polymerase chain reaction. We also used immunohistochemistry to examine the expression of hepatic TIPE2 protein in CHB patients.Results1. TIPE2, IL-6, IL-10, TNF-a, and IFN-y mRNA levels in chronic hepatitis B patients were significantly higher than healthy controls.2. TIPE2 mRNA level in immune clearance (IC) phases was significantly higher than immune tolerance (IT) phase (P<0.05); whereas TIPE2 mRNA in HBeAg negative hepatitis (ENH) was obviously higher than low replication (LR) phase(P<0.05 XThere was not find significant differences of TIPE2 mRNA in ENH and IC phases (P>0.05), as well as LR and IT phases (P>0.05).3. The level of IL-10 in IC group (3.33 [3.12,4.65]) was significantly higher than that in LR group (3.03 [2.79,3.39], P<0.05) or IT group (3.11 [0.31,3.27], P<0.05), whereas we also observe significantly difference of IL-10 between ITgroup and ENH group (3.11 [0.31,3.27] vs.3.37 [3.08,3.71], P< 0.05). However, we did not find any significant differences of IL-6, TNF-a and IFN-γ within any two of the four phases using Kruskal-Wallis analysis.4. A cut off value of 2.02 and 1.59 for TIPE2 mRNA level has significant power in discriminating IC from IT, and ENH from LR.5. The expression of hepatic TIPE2 was associated with the inflmmatory grade and the firosis stage in CHB patients.6. Spearman ananlysis revealed that the relative expression of TIPE2 mRNA was positively correlated with ALT (r=0.300, P<0.001), AST (r=0.283, P<0.001), PTA r=0.218, P=0.002), IL-10 (r=0.147, P=0.036). whereas negatively correlated with IL-6 (r=-0.163, P<0.001), TNF-a (r=-0.159, P=0.023), and IFN-γ (r=0.147, P=0.036).ConclusionsThe expression of TIPE2 was elevated in CHB patinents at gene and protein level. TIPE2 might contribute to immune phases in different stages of chronic HBV infection.Part Ⅲ:TIPE2 mRNA level in PBMCs serves as a novel biomarker for predicting short-term mortality of acute-on-chronic hepatitis B liver failureBackgoundsAcute-on-chronic liver failure (ACLF) is a severe life-threatening clinical syndrome, which is characterized by acute deterioration of liver function during the progression of diagnosed or undiagnosed chronic liver disease. In China, hepatitis B virus (HBV) associated ACLF, also termed as acute-on-chronic hepatitis B liver failure (ACHBLF), accounts for over 80% of ACLF. ACHBLF has a rapidly progressive course and may lead to a high short-term mortality.However; there are no specific and powerful regimes for the treatment of ACHBLF. Liver transplantation is the definitive treatment to lower mortality rate, but there is a great imbalance between donation and potential recipients. Agreeable consensus is that early identification of patients with high mortality and timely intervention would be effective for the treatment of ACHBLF. Several independent risk factors for 3-mortality of ACLF have been identified in consecutive cohorts, including serum total bilirubin(TBIL), international normalized ratio(INR), model for end-stage liver disease (MELD) and pharmaceutical treatment regimen. MELD was the most common used predictor for the severity of ACLF. Nevertheless, most of the predictive models are not HBV-specific to liver failure and usually have poor predictive value for ACHBLF. Therefore, new potential biomarkers are urgently needed to predict accurately the short-term prognosis of patients at the early stage of ACHBLF patients.Tumor necrosis factor-a-induced protein 8-like 2(TIPE2) is a newly identified negative regulatory molecular of immune response, and governs immune homeostasis by negatively regulating signaling through T cell receptors and Toll-like receptors.In the patients with ACHBLF, we also previously reported the elevation of TIPE2 mRNA and speculated that TIPE2 might negatively regulate cell-mediating immunity to abnormal liver function in a preliminary study. Therefore, we forward the hypothesis that TIPE2 mRNA might be a useful biomarker for predicting the outcome of ACHBLF patients.Aims1. To detect the expression of TIPE2 mRNA in PBMCs of ACHBLF patients, as well as its association with severity ofACHBLF2. To evaluate the diagnosis value of TIPE2 mRNA in peripheral blood mononuclear cells (PBMCs) for predicting 3-month mortality ofACHBLF patientMethodsThis prospective study consisted of 108 ACHBLF patients from March 2009 to May 2013 as training cohort and 63 ACHBLF patients from June 2013 to December 2014 as validation cohort.42 patients with chronic hepatitis B (CHB) and 22 healthy volunteers were also included as controls. The mRNA level of TIPE2 in PBMCs was determined using quantitative real-time polymerase chain reaction. Univariate analysis and Cox proportional hazard regression analysis were performed to identify independent risk factors to 3-monthmortality. Area under the receptor operating characteristic curve (AUROC) was performed to assess diagnostic value of TIPE2 mRNA in training and validation cohort.Results1. A total of 171 patients with ACHBLF,42 patients with CHB and 22 HCs were enrolled in this present study from March 2009 to December 2014. Of all the 171 ACHBLF patients,108ACHBLF patients hospitalized from March 2009 to May 2013 were set as training cohort; meanwhile,63 ACHBLF patients hospitalized from June 2013 to December 2014 were set as validating cohort. In the training cohort,67 patients died at the end of 3-month follow up and the mortality was 62%. Cirrhosis, hepatic encephalopathy (HE), ascites and spontaneous bacterial peritonitis were found in 59 (54.6%),39 (36.1%),50 (46.3%), and 40 (37%) patients with ACHBLF, respectively.2. The level of TIPE2 mRNA was significantly higher in ACHBLF patients (median (interquartile):6.5 [3.7,9.6]) compared with CHB (2.3 [1.6,3.7]) and healthy controls (0.4 [0.3,0.6]; both P<0.05). The expression of TIPE2mRNA was significantly positively correlated with MELD score (r= 0.544, P< 0.001,), HBsAg (r=0.229, P<0.05), and negatively correlated with PLT(r=-0.262, P<0.05). No significant correlation has been observed between TIPE2 mRNA and age (r= 0.141, P-0.146), ALT (r=-0.035, P=0.718), AST (r=-0.069, P= 0.475), ALB (r=-0.191, P=0.053), WBC (r= 0.185, P=0.055), and HGB (r= 0.033, P=0.734).3. Univariate cox proportional hazards regression analysisshowed that ALB (HR= 0.948,95% CI:0.906-0.993, P< 0.05), WBC (HR= 1.045,95% CI:1.015-1.076, P=0.003), cirrhosis (HR=1.977,95%CI:1.199-3.263, P=0.008), SBP (HR=3.052, 95%CI:1.875-4.967, P<0.001), ascites (HR=1.855,95% CI:1.141-3.017,P< 0.05), HE (HR=3.353,95%CI:2.062-5.453, P< 0.001), MELD score (HR= 1.093,95%CI: 1.052-1.136, P<0.001) and TIPE2 mRNA (HR= 1.202,95% CI:1.140-1.267, P<0.001)were significantly associated with mortality in the training cohort.4. Cox proportional hazards regression analyses showed 5 independent risk factors associated with 3-month mortality of ACHBLF:white blood cells (HR=1.058,95%CI: 1.023-1.095), spontaneous bacterial peritonitis (HR=2.541,95%CI:1.378-4.686), hepatic encephalopathy (HR=1.848,95%CI:1.028-3.321), model for end stage liver diseases (MELD) score (HR= 1.062,95%CI:1.009-1.118), and TIPE2mRNA (HR= 1.081,95%CI:1.009-1.159).5. An optimal cut-off point 6.54 of TIPE2 mRNA showed sensitivity of 74.63%, specificity of 90.24%, positive predictive value of 92.5% and negative predictive value of 67.3% for predicting 3-monthmortality in training cohort.6. TIPE2 mRNA plusMELD performed better than MELD alone for predicting 3 months mortality intraining (AUROC,0.853 vs.0.722, P<0.05) and validation cohort (AUROC,0.909 vs.0.717, P<0.001).ConclusionTIPE2 mRNA level might be a novel biomarker in predicting 3-month mortality of ACHBLF. Combination of TIPE2 mRNA and MELD would improve the diagnostic value of MELD alone in predicting 3-month mortality of patients with ACHBLF.